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05 March 2010, Volume 14 Issue 10 Previous Issue    Next Issue

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Differential expression of microRNA-125b in the neuronal differentiation of adipose-derived Flk1+ mesenchymal stem cells Wang Shi-hua, Bian Chun-jing, Huang Shan, Zhao Chun-hua 2010, 14 (10):  1711-1715.  doi: 10.3969/j.issn.1673-8225.2010.10.001 Abstract ( 323 )   PDF (471KB) ( 468 )   Save BACKGROUND: The differentiation of mesenchymal stem cells (MSCs) is strictly regulated by both genetic and epigenetic factors. Emerging evidences have demonstrated that microRNA also plays an important role in this process. OBJECTIVE: To explore the differential expression of microRNA-125b during neuronal differentiation of Flk1+ adipose-derived MSCs (AD-MSCs). MATERIALS：The fat samples were provided by healthy female volunteers aged 15-35 years and recruited from the Plastic Surgery Hospital affiliated to the Chinese Academy of Medical Sciences. METHODS: Adult adipose tissues were obtained from healthy volunteers with age of 15-35 years. Using adherence method, Flk1+ MSCs were obtained and the 3rd passage cells were taken in the experiment. Cultured in neuronal induction medium, these MSC were induced to differentiate towards neuronal lineage. The expression of microRNA-125b was examined at days 0, 4, 8 and 12. To explore its role in neuronal differentiation, we need to change its expression. RT-PCR and Taqman real-time PCR were carried out to explore the differentially expression of microRNA-125b during neuronal differentiation of AD-MSCs. The effect of inhibitor on the expression of microRNA-125b was detected. RESULTS AND CONCLUTION: ①The Flk-1+ MSCs were successfully induced into neuronal differentiation and displayed typical morphological changes 12 days after induction: Most cells retracted their cytoplasm, forming spherical cell body and emitted cellular protrusions. RT-PCR and immunocytochemistry studies confirmed their phenotype with expression of known neuronal cell markers including neurofilament, glial fibrillary acidic protein. ②The expression of microRNA-125b was significant up-regulated during neuronal differentiation. Results of RT-PCR and Taqman real-time PCR were concordance with that of microRNA chip technology. ③Inhibitor could down-regulate microRNA-125b. The results implied that microRNA-125b may play an important role in neuronal differentiation. Related Articles | Metrics

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Iodine effect on survival time of neuron-like cells differentiated from bone marrow mesenchymal stem cells Yan Wen-zhu, Qin Shu-jian, Liu Xue-zheng, Li De-hua 2010, 14 (10):  1716-1720.  doi: 10.3969/j.issn.1673-8225.2010.10.002 Abstract ( 383 )   PDF (460KB) ( 392 )   Save BACKGROUND: In vitro experiment has shown that the survival time of conventional chemical induction-induced neuron-like cells differentiated from bone marrow mesenchymal stem cells (BMSCs) was short, which limited its further application. With regard to the possibility of extension of iodine-induced neuron-like cells, the survival time has not yet been professionally reported. OBJECTIVE: To research the effects of the micro-element iodine on the survival time of neuron-like cells differentiated by BMSCs. METHODS: Rat mesenchymal stem cells at passage 3 were obtained under sterile condition, and divided into groups A-F. In group A, iodine ion was not added. In groups B-F, iodine ion at mass concentrations of 2, 55, 90, 125 and 2 500 mg/L was added respectively. An additional blank control group was established, and simultaneously the cells were induced into neuron-like cells with dimethyl sulphoxide (DMSO). Cells following induction were subjected to immunohistochemistry. Survival time of neuron-like cells was observed under different mass concentrations of iodine ion. RESULTS AND CONCLUSION: When mass concentrations of iodine ion were between 55 - 125 mg/L, the survival time of neuron-like cells prolonged to about 5 days and structures of induced cells were intact. From then on, the number of dead cells was gradually increased till approximately one week, all neuron-like cells died. When mass concentrations of iodine ion were 2 mg/L and 2 500 mg/L, cell survival time was from 12-36 hours. No significant difference was determined compared with group A. Till 2 or 3 days, all neuron-like cells died. Above-described results indicated that an appropriated concentration of iodine iron added in the common chemical induction may be benefit for the survival time of the neuron-like cells differentiated by BMSCs, but the effect may be negligible for the survival time of neuron-like cells induced when the added concentration of iodine iron is too low or too high. Related Articles | Metrics

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Coculture of human umbilical cord mesenchymal stem cells from Wharton’s jelly and brain tumor stem cells Tian Yi, Guan Fang-xia, Hu Xiang, Yang Bo, Du Ying, Zhou Chang-hui, Ba Yun-tao, Gu Chen-xi, Lei Ning-jing, Wang Xiao-wei 2010, 14 (10):  1721-1728.  doi: 10.3969/j.issn.1673-8225.2010.10.003 Abstract ( 333 )   PDF (769KB) ( 403 )   Save BACKGROUND: Human mesenchymal stem cells derived from Wharton’s jelly (WJCs) display the characteristics of MSCs as defined by the International Society for Cellular Therapy. They can be differentiated into bone, cartilage, adipose, muscle, and neural cells. They can also support the expansion of other stem cells, be well-tolerated by the immune system, and have the ability to home to tumors. OBJECTIVE: To investigate biological changes of WJCs and brain tumor stem cells (BTSCs) co-cultured in vitro. METHODS: WJCs cultured by situ cultivation and BTSCs used enzyme digestion way respectively, and gathering the 3rd passage of WJCs though subculturing as well as BTSCs. Two kinds of cells co-cultured in 24-well plates in serum-free medium (SFM) without any growth factor. 3 and 7 days after co-cultured respectively, CD133 expression of suspension cells in the 24-well plates were identified by flow cytometry, and immunofluorescence was performed for Nestin and glial fibrillary acidic protein (GFAP) expression of adherent cells. Co-culture supernatant (CCS) re-suspended 3rd passage of BTSCs and cultured into 96-well plates at day 3, which were used to determine the difference in cell growth curve in both groups using a microplate reader. RESULTS AND CONCLUSION: With the cocultivation days increasing, the phenomenon that tumor sphere cells began to be decomposed, adherent and differentiated observed by an inverted microscope. BTSCs in the co-cultured group expressed GFAP and Nestin when adherent and differentiated. The higher degree of malignant brain tumor tissue used in culturing BTSCs was, the higher expression of CD133 in BTSCs was. CD133+ in BTSCs declined when co-cultured with WJCs. Growth curve of brain tumor stem cells cultured in CCS compared with in SFM at day 3, which indicates that the proliferation of BTSCs inhibited obviously. Results indicated that CD133+ expression and proliferative capacity of BTSCs went down and BTSCs underwent differentiation during the co-culture in vitro. Related Articles | Metrics

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Biological characteristics of human umbilical cord mesenchymal stem cells following cryopreservation Wang You-wei, Han Zhi-bo, Yan Shu-lin, Mao Ai-bin, Wang Bin, Wang Ding, Chen Ke, Han Zhong-chao 2010, 14 (10):  1729-1733.  doi: 10.3969/j.issn.1673-8225.2010.10.004 Abstract ( 363 )   PDF (608KB) ( 630 )   Save BACKGROUND: An effective freezing-thawing technique is crucial for the clinical application of human umbilical cord mesenchymal stem cells (UC-MSCs). OBJECTIVE: To investigate biological characteristics of UC-MSCs after cryopreservation. METHODS: UC-MSCs were isolated from human umbilical cord and frozen in liquid nitrogen. The survival rate and the suppressive effect of γ-interferon (IFN-γ) of cryopreserved-thawed and fresh human UC-MSCs were compared. Furthermore, the multiple potentials and phenotype of UC-MSCs were estimated after cryopreservation. RESULTS AND CONCLUSION: There was no significant difference between cryopreserved-thawed and fresh human UC-MSCs on the survival rate and the suppressive effect of IFN-γ of peripheral blood mononuclear cells (PBMCs). After cryopreservation, human UC-MSCs had the potential differentiation and the phenotype of mesenchymal stem cells. Related Articles | Metrics

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Isolation, culture and differentiation of mesenchymal stem cells from Wharton’s jelly of human umbilical cord Jiang Jie, Tan Can, Zhang Li-yang, Xiao Ling, Zhang Jian-xiang 2010, 14 (10):  1734-1738.  doi: 10.3969/j.issn.1673-8225.2010.10.005 Abstract ( 341 )   PDF (505KB) ( 589 )   Save BACKGROUND: Bone marrow is the main source of mesenchymal stem cells (MSCs) at present, but its application has been limited, because of some reasons such as inconvenience of isolation, and the quantity of cells decreases with human increased age. Umbilical cord as a new source of MSCs has been widespread concerned recently. OBJECTIVE: To explore the approach of isolating and culturing MSCs from Wharton's jelly of human umbilical cord, and the methods of identifying the surface antigens and the differentiation potential. METHODS: MSCs were isolated and amplified via tissue-cultivation, and cultured by FasGrow medium. Morphology of MSCs from Wharton's jelly of human umbilical cord was observed under the optical microscope. Its immunophenotypes were detected using immunohistochemistry. The differentiation of MSCs into the osteoblasts was determined utilizing Gomori calcium-cobalt alkaline phosphatase staining, von Kossa calcium node staining, and tetracyclinefluorescence labeling. The differentiation of MSCs into the adipocytes was detected using oil red O staining. RESULTS AND CONCLUSION: MSCs were easily obtained from Wharton’s jelly of human umbilical cord via the proposed approach. The primary cells grew up to 70%-80% confluence after 12-16 days of culture, and meanwhile the undifferentiated state was maintained and proliferation was stabilized after passage. The cell cycle of double increase was about 2 days, and proliferation in vitro reached twenty generation above. Surface antigen analysis showed that CD44, CD105, CD133, MHC-Ⅰwere positively expressed, while CD34, CD45 were negatively expressed. Experiments of differentiation in vitro indicated that the obtained cells were capable of differentiating into fat, osteoblast and nerve-like cells Related Articles | Metrics

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Isolation, cultivation and reproductive activity of human umbilical cord blood mesenchymal stem cells Yan Man, Yang Yi-yong, Qin Shu-jian, Zheng De-yu 2010, 14 (10):  1739-1742.  doi: 10.3969/j.issn.1673-8225.2010.10.006 Abstract ( 409 )   PDF (411KB) ( 420 )   Save BACKGROUND: With increased age, bone marrow mesenchymal stem cells (BMSCs) are influenced with regard to quantity and quality, which will induce great damages to the donors. Many studies have focused on seeking substitute MSC source. In contrast, it remains controversial whether umbilical cord blood contains MSCs. OBJECTIVE: To isolate MSCs from human umbilical cord blood, and to detect their biological properties. METHODS: Umbilical cord blood samples were sterilely isolated using Percoll density gradient centrifugation to harvest intermediate layer cells. DMEM medium containing fetal bovine serum, penicillin, streptomycin and L-glutamine was added. Following several adherences and purification, the floating cells were discarded. Thus, many adherent cells with a confluence were collected. When cells were 60%-80% confluent, cells were digested by trypsin for subculture. Cells at passages, 1, 5 and 9 were obtained and their morphological changes were observed. Cell surface antigens were measured using flow cytometry. Growth curves were drawn, and cell viability was determined utilizing MTT. RESULTS AND CONCLUSION: Isolated umbilical cord blood MSCs presented an even size, showing spindle or star-shape fibroblasts-like cells. Umbilical cord blood MSCs at 1, 5, 9 passages were greatly positive for CD29, CD105 and CD166, but weakly positive for CD34 and CD45. Following 5 days of incubation, cells entered logarithmic growth phase. The number was decreased at day 9. Population doubling time was (53.5±8.32) hours. Cells grew well. Cells at 1-7 passages showed similar viability (P > 0.05). Till passage 9, cell proliferation viability was decreased, but no significant difference was determined (P > 0.05). Results verified that MSCs can be successfully isolated from umbilical cord blood in vitro. Cells at passages 1-9 presented a good reproductive activity. Related Articles | Metrics

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Isolation and biological characteristics of rat umbilical cord mesenchymal stem cells Liu Kui-li, Shi Bing-yi, Liu De-zhong, Jin Jian-gang, Li Hai-bin, Shi Ying-chang, Feng Kai, Xiao Li 2010, 14 (10):  1743-1748.  doi: 10.3969/j.issn.1673-8225.2010.10.007 Abstract ( 388 )   PDF (574KB) ( 431 )   Save BACKGROUND: There are many studies concerning rat bone marrow mesenchymal stem cells for immune tolerance following transplantation and tissue repair. However, there are no reports on umbilical cord mesenchymal stem cells (UCMSCs). OBJECTIVE: To establish a method of separating mesenchymal stem cells (MSCs) from rat umbilical cord, and to study its biological characteristics. METHODS: MSCs were separated from rat umbilical cord with enzyme method and tissue mass method, and then incubated in DMEM-LG medium. Cell morphology was observed under an inverted microscope. Growth curves of cells were drawn using cell counting. Cell cycle and surface antigen were detected with flow cytometry. Adipogenic differentiation and osteogenic differentiation were tested by immunohistochemistry. RESULTS AND CONCLUSION: Both of the two methods could obtain plenty of MSCs from rat umbilical cord. Primary culture showed that the efficiency of enzyme method was higher than tissue mass method. Passage time of the former was about 10 days and the latter was 14 days. The passage time of latter except primary culture was the same. Immunophenotype analysis showed that MSCs from rat umbilical cord expressed adhesion molecule and stromal cell markers, CD90 and CD106, but did not express hematopoietic cell markers, CD34 and CD45. In vitro induction test verified that rat UCMSCs have the potentials of adipogenic and osteogenic differentiation. Related Articles | Metrics

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Regulating effects of Wnt signaling pathway on differentiation of bone marrow mesenchymal stem cells into osteoblasts and adipocytes Li Ping-hua, Liu Yu-yu, Cui Liao 2010, 14 (10):  1749-1754.  doi: 10.3969/j.issn.1673-8225.2010.10.008 Abstract ( 546 )   PDF (447KB) ( 807 )   Save BACKGROUND: Disequilibrium of proportion of adipogenesis and osteogenesis from bone marrow mesenchymal stem cells (BMSCs) is associated with many bone diseases. However, it has been demonstrated that Wnt signaling pathway could play an important role in regulation of BMSC differentiation. OBJECTIVE: To investigate the different gene expression profiles and to find the target gene on Wnt signaling pathway of the BMSCs, after being induced to osteoblasts and adipocytes respectively using Wnt signaling pathway PCR array. METHODS: The third-passage BMSCs, after being induced to osteoblasts and adipocytes respectively for 7 days. The total mRNA of MSCs was extracted by Trizol. BMSC morphology was observed following osteogenic and adipogenic induction under an inverted microscope. Gene array was detected by rat Wnt signaling pathway PCR array. Non-induction group served as controls. The ratio of increase/reduction gene of osteoblasts and adipocytes was calculated. RESULTS AND CONCLUSION: Under an inverted microscope, BMSCs with high homogenicity were obtained following passage 3. BMSCs differentiated into osteoblasts following osteogenic induction, and into adipocytes following adipogenic induction. Compared with non-induction group, fifteen genes (Dkk1, kremen, FZD1, FZD7, et al.) were expressed up-regulated (ratio > 2) and 16 (sFrp 5, β-catenin, Dvl3, Tcf7, et al.) genes down-regulated (ratio < 0.5) when the third-passage BMSCs were induced to adipocytes. Six genes (Dkk1, kremen, β-catenin, Wnt11, et al.) were expressed up-regulated and 15 genes (sFrp5, sFRP4, Fzd1, et al.) down-regulated when BMSCs being induced to osteoblasts. Above-mentioned results suggested that Wnt signaling pathway plays an important role in the osteoblast and adipocyte differentiation from BMSCs. Related Articles | Metrics

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Paracrine effects of bone marrow mesenchymal stem cells on the apoptosis of adriamycin-injured cardiomyocytes: How to verify the inhibitory effects? 2010, 14 (10):  1755-1759.  doi: 10.3969/j.issn.1673-8225.2010.10.009 Abstract ( 401 )   PDF (445KB) ( 426 )   Save BACKGROUND: Several studies have shown the improvement of heart function through the introduction of mesenchymal stem cells(MSCs) from bone marrow, which may be attributed to secretion of various cytokines that accelerate endogenous reparative process, but not regeneration of cardiomyocytes. OBJECTIVE: To observe the anti-apoptotic effects of MSCs on adriamycin(ADR)- injured cardiomyocytes apoptosis in vitro through paracrine pathway. METHODS: In vitro cultured neonatal rat cardiomyocytes (3×108/L) were incubated into a 6-well plate, 3 mL/well. 72 hours later, these cells were assigned into 3 groups. The primary cultured neonatal rat cardiomyocytes in the ADR-injured and coculture groups were exposed to 1 mg/L ADR for 4 hours to establish experimental models of toxic cardiomyocytes. The normal control group was left intact. In the coculture group, rat bone marrow MSCs (BMSCs) at passage 3 were regulated to 3×108/L, 3 mL/well was added into Millicell device for 24 hours. Following model induction, the Millicell device was inseted into above-mentioned 6-well plate to establish coculture system. The levels of cytokines were measured in the conditioned medium from three cardiomyocytes groups. Effects of BMSCs on Caspase-9 and Caspase-3 activities, apoptosis and Bcl-2 and Bax protein expression in ADR-injured cardiomyocytes were measured. RESULTS AND CONCLUSION: Compared with the normal control group,the level of cytokine including insulin-like growth factor (IGF-1) and hepatocyte growth factor (HGF) was significantly higher in the medium from ADR-injured group,the activity of caspase-9, caspase-3 and the apoptosis rate increased significantly, the expression of Bax protein was higher and Bcl-2 Protein was lower in ADR-injured group(P < 0.05). Compared with the ADR-injured group, the level of IGF-1 and HGF in co-cultured group increased significantly, the apoptosis rate, Caspase-3 and Caspase-9 activities decreased significantly,the expression of Bax protein was lower while Bcl-2 Protein was higher than ADR-injured group (P < 0.05). Results indicated that BMSCs show increased cytokine secretion and significant anti-apoptotic effects on ADR-injured cardiomyocytes through paracrine pathway. Related Articles | Metrics

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Iodine effect on survival time of neuron-like cells differentiated from bone marrow mesenchymal stem cells Yan Wen-zhu, Qin Shu-jian, Liu Xue-zheng, Li De-hua 2010, 14 (10):  1760-1720.  doi: 10.3969/j.issn.1673-8225.2010.10.002 Abstract ( 373 )   PDF (460KB) ( 306 )   Save BACKGROUND: In vitro experiment has shown that the survival time of conventional chemical induction-induced neuron-like cells differentiated from bone marrow mesenchymal stem cells (BMSCs) was short, which limited its further application. With regard to the possibility of extension of iodine-induced neuron-like cells, the survival time has not yet been professionally reported. OBJECTIVE: To research the effects of the micro-element iodine on the survival time of neuron-like cells differentiated by BMSCs. METHODS: Rat mesenchymal stem cells at passage 3 were obtained under sterile condition, and divided into groups A-F. In group A, iodine ion was not added. In groups B-F, iodine ion at mass concentrations of 2, 55, 90, 125 and 2 500 mg/L was added respectively. An additional blank control group was established, and simultaneously the cells were induced into neuron-like cells with dimethyl sulphoxide (DMSO). Cells following induction were subjected to immunohistochemistry. Survival time of neuron-like cells was observed under different mass concentrations of iodine ion. RESULTS AND CONCLUSION: When mass concentrations of iodine ion were between 55 - 125 mg/L, the survival time of neuron-like cells prolonged to about 5 days and structures of induced cells were intact. From then on, the number of dead cells was gradually increased till approximately one week, all neuron-like cells died. When mass concentrations of iodine ion were 2 mg/L and 2 500 mg/L, cell survival time was from 12-36 hours. No significant difference was determined compared with group A. Till 2 or 3 days, all neuron-like cells died. Above-described results indicated that an appropriated concentration of iodine iron added in the common chemical induction may be benefit for the survival time of the neuron-like cells differentiated by BMSCs, but the effect may be negligible for the survival time of neuron-like cells induced when the added concentration of iodine iron is too low or too high. Related Articles | Metrics

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Influence of bone marrow mesenchymal stem cells on hepatic stellate cells proliferation: Regulation of Cyclin D1 and P27 expression Wang Dong-xu, Jiang Hai-xing, Su Si-biao, Qin Shan-yu, Liang Zi-yu 2010, 14 (10):  1764-1768.  doi: 10.3969/j.issn.1673-8225.2010.10.011 Abstract ( 463 )   PDF (389KB) ( 571 )   Save BACKGROUND: The hepatic stellate cells (HSCs) plays a key role in the development of liver fibrosis. Studies have shown that bone marrow-derived mesenchymal stem cell (BMSCs) transplantation can be used to treat liver fibrosis, but the mechanism for reversal of liver fibrosis remains unknown. OBJECTIVE: To explore the mechanism of bone marrow mesenchymal stem cells to regulate the proliferation of HSCs under co-culture in vitro. METHODS: Rat BMSCs and HSCs in the experimental group were cultured in the plastic culture plate (6 holes) to establish the upper and lower double-cell co-culture system. Rat normal fibroblast cell lines were seeded as control group; HSCs were cultured alone as blank group. Cell proliferation was determined by WST8 and cell cycle was determined by flow cytometry. The Cyclin D1 and P27 mRNA expression in HSC was determined by reverse transcription-polymerase chain reaction (RT-PCR) and the level of Cyclin D1 and P27 protein by Western blot. RESULTS AND CONCLUSION: HSCs co-cultured with BMSCs significantly inhibited HSC proliferation compared with the blank and control groups at 24, 48, and 72 hours (P < 0.01); Flow cytometry showed that the percentage of G0/G1 phase cells of co-culture group was increased but the S phase cells reduced (P < 0.01) compared with the other groups at 72 hours, and BMSCs blocked HSC to convert from G0/G1 period to S phase. After HSCs co-cultured with BMSCs for 24 hours, the expression of CyclinD1 mRNA and protein was reduced, and significantly less than the blank and control groups at 72 hours (P < 0.01); no differences were detected in P27 mRNA expression in each group during the co-culture (P > 0.05). After co-culture of 24 hours, the p27 protein expression was significantly increased compared with the blank and control groups (P < 0.01). BMSCs inhibited the proliferation of HSCs, possibly through inhibiting CyclinD1 expression, increasing the p27 protein expression to cause cell cycle arresting in G0/G1 phase. Related Articles | Metrics

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Rat bone marrow mesenchymal stem cells induce hepatic stellate cells apoptosis in vivo Lin Nan, Xie Shu-jie, Pan Wei-dong, Hu Kun-peng, Chen Si, Chong Yu-tian, Xiang Peng, Xu Rui-yun 2010, 14 (10):  1769-1774.  doi: 10.3969/j.issn.1673-8225.2010.10.012 Abstract ( 238 )   PDF (909KB) ( 452 )   Save BACKGROUND: It is reported that bone marrow mesenchymal stem cell (BMSC) transplantation might be a promising treatment for liver fibrosis. But the mechanism is still unclear. OBJECTIVE: To observe the hepatic stellate cells apoptosis induced by BMSC transplantation, and to study the mechanism of BMSC in treating hepatic fibrosis in vivo. METHODS: CCl4 subcutaneous injection was performed to induce rat liver fibrosis. After 8 weeks of CCl4 injection, 20 rats which underwent successful model establishment were randomly divided into experimental group and control group, 10 in each group. The experimental group received MSC transplantation via tail vein injection, and the control group were given DMEM instead. The rats were killed and the livers were harvested at three time point, the day of MSC transplantation, 3 days after transplantation, and 7 days after transplantation. The hydroxyproline content was detected by HE and Masson staining, and the expression changes of α-smooth muscle actin (α-SMA) proteins were determined using immunohistochemistry. The apoptosis of hepatic stellate cells were determined by α-SMA and TUNEL (terminal dUTP nick-end labeling) dual-staining. RESULTS AND CONCLUSION: After 8 weeks of CCl4 injection, the hydroxyproline content increased and histology indicated progress of liver fibrosis. At 7 days after MSC transplantation, the hydroxyproline in the liver was decreased, and the liver fibrosis was alleviated in the experimental group but aggravated in the control group. Immunohistochemistry indicated that α-SMA positive cells were increased at 8 weeks after CCl4 injection. At day 7 after transplantation, α-SMA positive cells in the experimental group were significantly less than control group (P < 0.05). At 3 days after transplantation, the hepatic stellate cells apoptosis in the experimental group was significantly aggravated compared with control group (P < 0.05). This suggested that MSC transplant was an effective treatment for liver fibrosis. MSC inducing hepatic stellate cells apoptosis may be one of the mechanisms. Related Articles | Metrics

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Effects of separation methods and culture conditions on biological characteristics of rabbit bone marrow mesenchymal stem cells Weng Xuan, Zhu Yong-jun, Zhang Jian, An Hong 2010, 14 (10):  1775-1779.  doi: 10.3969/j.issn.1673-8225.2010.10.013 Abstract ( 378 )   PDF (678KB) ( 471 )   Save BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) are widely utilized as seed cells or carriers in bone tissue engineering and gene therapy. Thus, how to obtain BMSCs with high purity arose more attentions of researchers. OBJECTIVE: To observe the effects of different separation methods and culture conditions on biological characteristics of rabbit BMSCs. METHODS: BMSCs were obtained by whole bone marrow culture, density grand centrifugal and red blood cell lysis. At 48 hours after culture, the cell numbers were counted, the time of passage was recorded, in addition, the cell morphology was observed by phase contrast microscope, and the CD44 antigen expression was identified using flow cytometry. The 3rd and the 7th generation aging cells were cultured with DMEM-LG, MEM-HG, and DMEM/F12 culture medium. MTT and count cell plat were used to evaluate the growth of BMSCs. Phase contrast microscope was used to observe the morphological changes of aging cells. RESULTS AND CONCLUSION: BMSCs could be separated by each method. The adherent cells showed shuttle or multiple angle shapes, with rich cytoplasm, and positive for CD44 antigen. The more cell number and shortest primary culture time was presented in red blood cell lysis group (P < 0.05). DMEM/F12 could promote the proliferation of quiescent cells. And the cells prevented the better viability. The method of read blood cell lysis improving the efficiency of BMSCs adherent is an effective method of extraction of BMSCs. DMEM/F12 could promote the proliferation Maybe, DMEM/F12 is more suitable for BMSCs. Related Articles | Metrics

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Screening of optimal embryonic time for in vitro separation and culture of Kunming mouse embryonic stem cells: Comparison among 2.5, 3.5, and 4.5 pregnant days Zhang Hai-feng, Shan Wei, Qin Shu-jian 2010, 14 (10):  1780-1784.  doi: 10.3969/j.issn.1673-8225.2010.10.014 Abstract ( 496 )   PDF (450KB) ( 383 )   Save BACKGROUND: Genotype of Kunming mice was similar to human population, thus an establishment of embryonic stem cell line is beneficial for research of transgenic animal. However, the best time to collect embryo has been less reported yet. OBJECTIVE: To find the best time to collect embryos from Kunming mice. METHODS: The embryos were collected from mother mice of 2.5, 3.5, and 4.5 pregnant days. Microscope was used to evaluate the growth condition of embryos, embryo attaching rate (A/C), inner cell mass (ICM) growing rate (I/C), embryonic stem cells (ESCs) clone growing rate (P1/C) and ESCs subclone growing rate (P2/C). The cells were then stained with alkaline phosphatase. RESULTS AND CONCLUSION: Most of the 2.5-pregnant-day embryos were 16-cell-phase embryos. The 3.5-pregnant-day embryos were morulas while the 4.5-pregnant-day embryos were blastulas. There were no significant differences in A/C, I/C, P1/C, P2/C between 2.5 and 3.5 pregnant days (P > 0.05). The 4.5-pregnant-day indicators mentioned above were significantly greater than those two groups; therefore, 4.5-pregnant-day embryos were the best source to culture, clone, isolate and passage ESCs. Related Articles | Metrics

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Insulin-like growth factor-1 effects on directional differentiation of human adipose-derived mesenchymal stem cells into chondrocytes Zhou Quan, Deng Zhan-sheng, Zhu Yong, Li Bao-jun, Zhang Shao-xian, Zhao Jia-li 2010, 14 (10):  1785-1790.  doi: 10.3969/j.issn.1673-8225.2010.10.015 Abstract ( 252 )   PDF (503KB) ( 408 )   Save BACKGROUND: Recently, researches have found that insulin-like growth factor-1 (IGF-1) can induce the differentiation of bone marrow-derived mesenchymal stem cells (BMSCs) into chondrocytes, but there are no reports concerning the differentiation of adipose-derived mesenchymal stem cells (ADMSCs) into chondrocytes induced by IGF-1, as well as interaction with transforming growth factor-β1 (TGF-β1) during this process.  OBJECTIVE: To explore the possibility of inducing ADMSCs chondrogenic differentiation by using IGF-1 and the interaction with TGF-β1 in induction. METHODS: ADMSCs were obtained, and seeded at 2×105cells/cm2 in culture flask. Insulin-free chondrogenic media containing IGF-1 or (and) TGF-β1 were used to induce ADMSCs. 2 weeks later, cells were harvested and stained by using toluidine blue and collagen Ⅱ antibody immunohistochemistry. Intracellular sulfated proteoglycan and collagen Ⅱ coloring were observed. Reverse transcription-polymerase chain reaction (RT-PCR) was used to detect the expression of collagen Ⅱ, aggrecan and Sox9 mRNA. RESULTS AND CONCLUSION: After induced, toluidine blue stain exhibited that the cells in the three induction groups were polygonal, with cytoplasm and cell membrane of blue different dyeing. Immunohistochemistry for type II collagen demonstrated that cytoplasm and cell membrane were stained brown in three induction groups. RT-PCR revealed that the expression of collagen Ⅱ, aggrecan, Sox9 mRNA of IGF + TGF group were significantly greater than the IGF and TGF groups, and IGF and TGF groups were significantly stronger than the control group. No significant difference was determined between the IGF and TGF groups. These results indicated that IGF-1 can induce chondrogenic differentiation from ADMSCs, expressing chondrocyte specific cell phenotype. There is synergism of IGF-1 and TGF-β1 to induce the differentiation of ADMSCs into chondrocytes. Related Articles | Metrics

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Vitamin A effects on growth and proliferation of mouse spermatogonial stem cells during in vitro culture Hu Jian-xin, Sun Zhao-lin, He Jian, Liang Shao-feng, Zhang Yong, Tan Zong-jian, Yuan Jun 2010, 14 (10):  1791-1794.  doi: 10.3969/j.issn.1673-8225.2010.10.016 Abstract ( 425 )   PDF (485KB) ( 448 )   Save BACKGROUND: Vitamin A has important effects on growth of spermatogonial stem cells. At present, we have not found an inductive substance of promoting growth and differentiation of spermatogonial stem cells during in vitro culture. OBJECTIVE: To investigate the effect of Vitamin A on growth and proliferation of mouse spermatogonial stem cells in vitro culture. METHODS: Bilateral testis of Kunming male mice aged 5-7 days were sterilely collected. Spermatogonial stem cells were isolated and purified using adherence and noncontinuity Percoll density gradient centrifugation. Bilateral testis was obtained from Kunming male mice aged 12-15 days under sterile conditions. Sertoli cells were isolated and purified by using enzyme digestion method. Following adherence and polarization, Sertoli cells served as feeder layer. The spermatogonial stem cells were seeded on simple-layer Sertoli cells. We set two groups, In the experimental group, 1 g/L Vitamin A was added in the DMEM/F12. In the control group, Vitamin A was not added. Growth and proliferation of spermatogonial stem cells were measured using enzyme linked immunosorbent assay. The cell cycle of spermatogonial stem cells was determined by flow cytometry. RESULTS AND CONCLUSION: At days 6, 9, 12 and 15 following coculture, absorbance value of spermatogonial stem cells in experimental group was faster than control group (P < 0.05 or 0.01). With prolongation of coculture, quantity of chromosome in S phase in spermatogonial stem cells was increased, and then decreased in the experimental group. Another division cycle began. Compared with experimental group, quantity of chromosome in S phase in spermatogonial stem cells was slowly increased in the control group (P < 0.05). During in vitro culture of mouse spermatogonial stem cells, Vitamin A can improve the proliferation and polarization of spermatogonial stem cells. Related Articles | Metrics

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Screening and identification of oval cells-like stem cells of Thy-1-labeled positive hepatoma carcinoma cells: Are they tumor stem cells?   Cheng Bian-qiao, Jiang Yi 2010, 14 (10):  1795-1798.  doi: 10.3969/j.issn.1673-8225.2010.10.017 Abstract ( 338 )   PDF (398KB) ( 446 )   Save BACKGROUND: There may be tumor stem cells during metastasis and recurrence of liver cancer. The number of tumor stem cells is very few, so it is difficult to be identified. Thy-1 is a surface marking protein of oval cells. Specific marker can indirectly confirm the existence of tumor stem cells in the tumor, which is a focus in recent studies. OBJECTIVE: To study whether Thy-1 positive marked oval cells was expressed in hepatoma carcinoma cells, and investigate its screening and identification. METHODS: Flow cytometry was used to identify that Thy-1 positive expression in hepatoma carcinoma cells HepG2 and BEL-7402 and normal hepatocytes QSG-7701. Immunomagnetic bead sorting was utilized to select hepatoma carcinoma cells with Thy-1. Immunofluorescence was used to identify selected efficiency. Naked mouse inoculated with hepatoma carcinoma cells in the skin test was assigned to three groups. In the Thy-1+ cells group, naked mice were inoculated with Thy-1+ cells (5×105/mouse) underneath the skin of the back. In the HepG2 cell positive control group, naked mice were inoculated with HepG2 cells (0.5×107/mouse) underneath the skin of the back. In the Thy-1- cells negative control group, naked mice were inoculated with Thy-1- cells (0.5×107/mouse) underneath the skin of the back. Tumorigenicity in each group was observed one month later. RESULTS AND CONCLUSION: Thy-1 expression in hepatoma carcinoma cells HepG2 and BEL7402 was significantly greater compared with normal hepatocytes QSG7701 (P < 0.05). Positive rate of immunofluorescence staining was 85% in the Thy-1+ cell group, 1% in the HepG2 cell positive control group, 0% in the Thy-1- cell negative control group. There were nubble of five mice in HepG2 cell positive control and Thy-1+ groups, and the volume was large. However, there was one nubble in Thy-1- group, and the volume was significantly small (F=144.568, P < 0.05). Results indicated that hepatoma carcinoma cells contained Thy-1 labeled positive oval cells-like stem cells, which may be tumor stem cells in hepatoma carcinoma cells. Related Articles | Metrics

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Olfactory ensheathing cells transplantation for brain injury: Feasibility analysis and effect validation Wang Guang-zhi, Liu Ming-na 2010, 14 (10):  1799-1802.  doi: 10.3969/j.issn.1673-8225.2010.10.018 Abstract ( 460 )   PDF (463KB) ( 447 )   Save BACKGROUND: Brain injury is a serious central nervous system trauma. However, it is difficult to promote nerve regeneration and functional recovery after brain injury. Sheath cells are conducive to neuronal survival and axonal regeneration. OBJECTIVE: To explore feasibility and effect of olfactory ensheathing cells transplantation on rat brain injury. METHODS: A total of 90 healthy adult male SD rats were selected and 10 were used to prepare olfactory ensheathing cells. The remaining were randomly divided into model control and transplantation groups with 40 animals in each group. Model of middle cerebral artery occlusion was established by thread method. At 1 week, 2 × 106 suspension of olfactory sheath cells and an equal volume of sterile saline were injected into two groups, respectively via the carotid artery. Neurological deficits were evaluated by creeping scores; histopathological changes were detected by HE staining, and glial fibrillary acidic protein and neurotrophic factor receptor p75 expression was detected by immunohistochemistry. RESULTS AND CONCLUSION: Compared with the model control group, the neurological deficit scores were significantly reduced in the transplantation group compared with the control group at 1, 2, 3, and 4 weeks following cerebral ischemia/reperfusion (P < 0.05); the pathological changes in injured brain tissues were ameliorated, the number of nerve cell degeneration and necrosis was significantly reduced, and edema was attenuated. A great amount of glial fibrillary acidic protein and neurotrophic factor receptor p75 expression was detected in the infarct hemisphere following cell transplantation, and little in the contralateral hemisphere and vascular endothelial cells. Negative expression was detected in the model control group. Results show that the olfactory ensheathing cell transplantation is effective on ischemic brain injury in the rats. Related Articles | Metrics

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Effects of neural stem cell transplantation on hippocampus synaptophysin expression and learning memory abilities of Alzheimer disease rats Yang Chun, Zhou Hui,Bai Lin-lin, Wang Shu-chun, Zhang Qian 2010, 14 (10):  1803-1807.  doi: 10.3969/j.issn.1673-8225.2010.10.019 Abstract ( 325 )   PDF (506KB) ( 421 )   Save BACKGROUND: Previous studies have demonstrated that transplanted neural stem cells can survive and proliferate in the brain of Alzheimer disease (AD) rats, however, it is poorly understood whether it can rebuild the nerve tracts by substituting the injured or dead neurons and improve learning and memory abilities. Synaptophysin is one of the important markers of synaptic rebuilding. OBJECTIVE: To observe the effects of neural stem cell transplantation on synaptophysin expression in hippocampus and learning and memory abilities of AD rats. METHODS: Sprague Dawley rats were randomly divided into the normal control, AD model, 2-week-transplantation and 4-week-transplantation groups. All rats were established AD models except that in the normal control group. Neural stem cells were isolated from the dentate gyrus of hippocampus of newborn rats, labeled with Hoechst33258, and then transplanted into CA1 region of hippocampus of rats in the 2-week-transplantation and 4-week-transplantation groups. The behavioral testing in the rats was performed using Y-maze trial. Nissl staining and synaptophysin immunohistochemistry were detected after the rats were sacrificed. The same volume of stroke-physiological saline solution was injected into rats in the AD models group using the identical methods. There was no treatment in the normal control group. RESULTS AND CONCLUSION: ①The cells number in the hippocampal CA1 region of the 2-week-transplantation and 4-week-transplantation groups were increased than that of AD model group, but were still less than that of the normal control group (P < 0.05). There was no significantly difference between the absorbance values of 2- or 4- week-transplantation group and control group (P > 0.05). ②The absorbance values of the 2-week-transplantation and 4-week-transplantation were significantly greater than that of the control and AD model groups (P < 0.05). ③The learning and memory abilities in 2- and 4-week-transplantation group enhanced obviously and their correct reaction rates improved evidently, which was found statistically significant difference from AD model group (P < 0.05), while no statistically significant difference from control group (P > 0.05). The transplanted neural stem cells may promote the synaptic rebuilding and improve learning and memory abilities in AD rats. Related Articles | Metrics

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Ca2+ signaling mediated salidrosides promotes directional differentiation of mouse bone marrow mesenchymal stem cells into nerve cells Pei Jing-jing, Wu Run, Zhao Hong-bin, Liu Xu-dong, Hu Jun, Bai Meng-hai 2010, 14 (10):  1808-1812.  doi: 10.3969/j.issn.1673-8225.2010.10.020 Abstract ( 294 )   PDF (451KB) ( 535 )   Save BACKGROUND: Tranditional Chinese medicine, which possesses anti-oxidation properties, can promote directional differentiation of mouse bone marrow mesenchymal stem cells (BMMSCs) into nerve cells. Salidrosides, as the effective constituent of Rhodiola, have strong anti-oxidation function. OBJECTIVE: To investigate the molecule mechanism of salidrosides induced differentiation of mouse BMMSCs into nerve cells. METHODS: When in vitro cultured BMMSCs reached 80% confluency, the cells were assigned into 3 groups. Cells in the control group were cultured by complete culture medium; those in the induction and positive control groups were cultured by complete culture medium adding 20 mg/L salidrosides or 0.1 mg/L nerve growth factors (NGF). The related gene and protein of nerve cells were detected using RT-PCR and Western blot method at 12 hours after culture. After that, the cells in the induction group were divided into 3 groups, the blocking agents EGTA (Ca2+ chelator), Nifedipine (L-type Ca2+ channel blocker) and LY294002 (IP3 receptor blocking pharmacon) were applied to block the cellular Ca2+ signal pathway respectively for 12 hours. RT-PCR and Western blot methods were used to study the signal transduction of the salidrosides. RESULTS AND CONCLUSION: ①The expression of neuron specific enolase (NSE), β-Tubulin III, Nurr1 mRNA could be found in the induction and positive control groups, instead of the control group; The expression abundance of the positive control group was smaller than that of the induction group. The expression abundance of GFAP mRNA was very low in each group, but the c-fos mRNA was expressed abundantly in the induction group. ②Compared with the positive control group, the induction group could promote the NSE expression obviously, which was no expressed in the blank control group. ③The expression of NSE and Nurr1 were conspicuously down-regulated when the Ecto Ca2+ and L-type Ca2+ channel and IP3 receptor were blocked respectively. Salidrosides can induce the differentiation BMMSCs into nerve cells. Ca2+ signaling and IP3 dependent Ca2+ signaling pathway play an important role in transduction salidrosides signal in BMMSCs differentiation. Related Articles | Metrics

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Effect of basic fibroblast growth factor on proliferation and differentiation of monkey bone marrow mesenchymal stem cells into neuronal precursor cells: Does the concentration affect cryptotanshinone induction? Liu Xiao-gang, Deng Yu-bin, Cai Hui 2010, 14 (10):  1813-1816.  doi: 10.3969/j.issn.1673-8225.2010.10.021 Abstract ( 254 )   PDF (465KB) ( 443 )   Save BACKGROUND: Basic fibroblast growth factor (bFGF) belongs to active peptide, which is an effective mitogenic factor. OBJECTIVE: To investigate the effect of bFGF on proliferation and differentiation of monkey bone marrow mesenchymal stem cells (BMMSCs) into neuronal precursor cells. METHODS: Monkey BMMSCs were in vitro cultured by density gradient centrifugation, and then divided into 4 groups after passaged, namely, control, bFGF with low, medium and high concentration groups. In the bFGF groups, 0, 3, 6, 10 μg/L bFGF were applied. The proliferation of BMMSCs in each group were observed. The 5th BMMSCs were cultured with serum free L-DMEM culture medium containing 20 mg/L cryptotanshinone to differentiated into neural-like cells. The expression of positive-nestin protein was detected by immunohistochemical method. RESULTS AND CONCLUSION: Compared with the control group, proliferation rate of BMMSCs in the bFGF groups were accelerated (P < 0.05), which showed a positive correlation to the concentration of bFGF. The positive-nestin protein could be found in the low and medium concentration groups at 0.5 hours after induction, and reached a peak at 1.5 hours, which increased obviously in the low concentration group than that of the high concentration group (P < 0.05). bFGF can promote BMMSCs proliferation in vitro, enhance inducing ratio of prophase neuron-like cells at lower concentration but inhibit differentiation at high level. Related Articles | Metrics

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Effect of Astragaloside IV on hematopoietic growth factor expression in rat bone marrow mesenchymal stem cells Tan Yan-fang, Yin Xiao-cheng, Xiong Yu-juan, Wang Yan 2010, 14 (10):  1817-1820.  doi: 10.3969/j.issn.1673-8225.2010.10.022 Abstract ( 313 )   PDF (428KB) ( 484 )   Save BACKGROUND: Astragaloside IV is a major component of Huangqi, promoting proliferation and differentiation of bone marrow mesenchymal stem cells; however, the mechanism has been less reported yet. OBJECTIVE: To explore the effect of Astragaloside IV on expression of multiple hematopoietic growth factors in bone marrow mesenchymal stem cells. METHODS: Bone marrow mesenchymal stem cells were isolated from adult Wistar rats by using the method of adhesive culture and clone, and they were then plated on 96-well plate and separately incubated with 100 μL Astragaloside IV (25, 50, 100, 200 g/L) for 72 hours. The cells in the control group were cultured with an equal volume of DMEM-LG culture liuquid. Indirect immunofluorescence was used to detect the biological activity, MTT method was used to evaluate the effect of Astragaloside IV on proliferation and differentiation of bone marrow mesenchymal stem cells, and RT-PCR method was used to measure the expression of hematopoietic growth factors in bone marrow mesenchymal stem cells. RESULTS AND CONCLUSION: The 3rd-passage bone marrow mesenchymal stem cells highly expressed CD44 but lowly expressed CD45. As compared with control group, Astragaloside IV promoted proliferation of bone marrow mesenchymal stem cells in a time/dosage-dependent manner, in particular, the 200 g/L Astragaloside IV and 72-hour intervention (P < 0.05). SCF expression was significantly increased in the drug group compared with control group (P < 0.01); however, TPO, GM-CSF, and TGF-β1 expressions were not changed significantly (P > 0.05). Moreover, interleukin-3 expression was not found in the bone marrow mesenchymal stem cells. Astragaloside IV promoted in vitro proliferation of bone marrow mesenchymal stem cells, possibly involving in SCF secretion. Related Articles | Metrics

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Detection and separation of side population cells in haematological tumor cell lines Fan Rui-hua, Li Hui-min, Li Xiao-jin, Yu Mei-jia, Jin Cong-guo 2010, 14 (10):  1821-1824.  doi: 10.3969/j.issn.1673-8225.2010.10.023 Abstract ( 370 )   PDF (385KB) ( 459 )   Save BACKGROUND: Side population (SP) cells, with varied contents, are widely distributed in adult tissues, embryos, and certain tumor cells. Haematological tumor is one of the main pathological conditions, which endangers human life. Thus, it is important to recognize SP cells in haematological tumor cell lines. OBJECTIVE: To identify whether hematologic tumor cell lines contain SP cells, and to explore a stable detection and separation methods. METHODS: Cells with the characteristics of stem cells being capable of fluorescent dye Hoechst33342 were isolated by flow cytometry; whether there were SP cells in logarithmic growth period of NB4, Raji, K562/ADM and K562 or not were detected. After sorting K562 subpopulations, the purity of sorted cells was detected.  RESULTS AND CONCLUSION:After Hoechst33342 staining, flow cytometry results showed that the SP cells appeared in the haematological tumor NB4, Raji, K562/ADM and K562 cell lines, which accounted for 0.8%, 2.7%, 1.3% and 2.7%, respectively. These cells could be blocked by Verapamil. The purity was greater after a second detection of SP and Non SP cells in K562 cells. Related Articles | Metrics

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Culture and preparation of dog bone marrow mesenchymal stem cell sheet in vitro Jing Heng, Tan Shuai, Gao Zhen-hua, Chen Li-qiang, Li Ning-yi 2010, 14 (10):  1825-1828.  doi: 10.3969/j.issn.1673-8225.2010.10.024 Abstract ( 289 )   PDF (448KB) ( 382 )   Save BACKGROUND: There are some disadvantages in harvesting and transferring cells in the traditional tissue engineering technique, and it is difficult to form dense tissues, which significantly limits the development of tissue engineering. OBJECTIVE: To explore the culture and fabrication of dog bone marrow mesenchymal stem cell (BMSC) sheet in vitro. METHODS: Bone marrow was extracted from dogs following anesthesia. BMSCs were isolated with the method of density gradient centrifugation in vitro. BMSCs at passage 4 at a density of 1×109/L were incubated in the temperature-responsive culture dishes with a diameter of 3.5 cm, and cultured in an incubator at 37 ℃, 5% CO2 and saturated humidity. The temperature of the incubator was changed from to 37 ℃ to 20 ℃ to prepare BMSCs cell sheet for 20 minutes. Cell morphological changes and cell sheet formation were observed under an inverted microscope. RESULTS AND CONCLUSION: Dog BMSCs following 24 hours of primary culture presented ellipse or polygonal shape. Most cells adhered at hour 72, and cell colonies were visible at day 7. Cells showed long spindle and completely confluence at day 12, with unclear boundary. BMSCs in the temperature-responsive culture dishes presented short spindle shape, and gradually separated from the dish bottom, forming entire cell sheet containing extracellular matrix at 20 ℃. These verified that dog BMSCs can be effectively obtained with method of density gradient centrifugation. Complete cell sheet layer can be fabricated with temperature-responsive culture dishes. Related Articles | Metrics

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Human Schwann cells from normal nervous tissue cultured in vitro Zhang Zhi-jun, Wang Shi-jie, Sun Zhao-hui, Ao Qiang, Li Yan, Liu Qiang 2010, 14 (10):  1829-1832.  doi: 10.3969/j.issn.1673-8225.2010.10.025 Abstract ( 209 )   PDF (424KB) ( 404 )   Save BACKGROUND: Peripheral nerve tissue engineering needs a large number of Schwann cells. In previous studies, lack of normal human nervous tissue, so animal (rat, rabbit, et al) nervous tissues are commonly used to isolate Schwann cells, but as xeno-cells it is limited in clinical application. OBJECTIVE: To investigate an effective technique for isolation, cultivation and purification of human Schwann cells of normal peripheral nerves cultured in vitro. METHODS: Normal peripheral nerves were obtained from the surgery of cerebral palsy patients. Schwann cells were cultured with enzymatic digestion culture method and differential attachment method. Tissues were cut into pieces and incubated in medium supplemented with fetal bovine serum, collagenase and Dispase enzyme, centrifuged. Tissue blocks were placed in the medium, triturated into monoplast suspension, and then moved into a DMEM Petri dish containing polylysine, supplemented with basic fibroblast growth factor. When adherent cells were confluent about 85%-90%, cells could subculture. Schwann cells were counted by Trypan Blue coloring method at 2, 3, 4, 5, 6, 7, 8, 9, 10. The purity of Schwann cells was identified through S-100 protein immunohistochemistry staining.  RESULTS AND CONCLUSION: More than 0.5×108/L Schwann cells were detected after four days under a microscope. Following the third passage, the number of Schwann cells was over 9×108/L. The purity of Schwann cell population was up to 85%. Results suggested that plenty and purified human Schwann cells could be obtained by enzymatic digestion culture and differential attachment methods in a short time, which can be used for the source of peripheral nerve tissue engineering. Related Articles | Metrics

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Isolation and purification of human cytotrophoblasts and placental mesenchymal stem cells Sha Wen-qiong, Wang Zi-neng, Wang Dong-ju 2010, 14 (10):  1833-1837.  doi: 10.3969/j.issn.1673-8225.2010.10.026 Abstract ( 332 )   PDF (518KB) ( 649 )   Save BACKGROUND: Cytotrophoblasts in placental cell components plays an important role in fetal immunological tolerance. Placental mesenchymal stem cells (pMSCs) have potential of multiple differentiation and inhibition of lymphocyte proliferation. However, conventional methods cannot acquire a large amount of purified human cytotrophoblasts or pMSCs. OBJECTIVE: To establish a method to obtain large placenta tissue, and harvest plenty of cytotrophoblasts and pMSCs with high purity and activity. METHODS: Human placenta tissues were dissected, minced, and dissociated in trypsin and DNAse I. The dissociation was performed in three stages of incubation at 180 r/min for 20 minutes at 37 ℃. The digesting suspension was filtered using a 200 mesh strainer before separated by Percoll gradients. The cytotrophoblast cells and pMSCs fractions were collected respectively. Fibroblasts of cytotrophoblast cells fraction were removed by differential adhesion. The pMSCs were seeds on 75-cm2 flask directly for culture. The dissociation of placenta tissue was observed. The number of harvesting cytotrophoblasts was quantified and Cytokeratin 7 expression was tested. The pMSCs primary culture time, cell passage, induced osteoblast differentiation were observed. The cell surface makers were also detected. RESULTS AND CONCLUSION: After digesting in trypsin and DNAse I, there was only little residue left. (5.48±1.98)×108 cytotrophoblasts were obtained after differential adhesion. (90±4.36)% of these cells were positive for Cytokeratin 7. At 19-21 days after pMSCs reached approximately 90% confluency, the cell number was (1.96±0.24)×106. The subcultre cells could be passaged again in 4 or 5 days. Flow cytometric analysis of pMSCs showed that the cells expressed CD29, CD44 and HLA-ABC intensively and were negative for CD34, CD45, CD14 and HLA-DR. pMSCs differentiated into osteoblast-like cells after induction, which stained bright salmon pink by Alizarin Red. Dissociating the placenta tissue in trypsin and DNAse Ⅰ in combination with discontinuous Percoll gradient separation obtained a large number of cytotrophoblasts and pMSCs recovered from placenta tissue, with high purity and activity. Related Articles | Metrics

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E-selectin expression, mouse embryonic development and hematopoietic function Xu Li-ping, Fang Fang, Xu He-biao, Ma Ning-fang 2010, 14 (10):  1838-1842.  doi: 10.3969/j.issn.1673-8225.2010.10.027 Abstract ( 562 )   PDF (702KB) ( 485 )   Save BACKGROUND: There are reports concerning effects of E-Selectin, a cellular adhesion molecule, on selectively adhesion among cells, regulation of leukocyte homing and exudation, and tumor cell transfer. However, there are no reports addressing E-Selectin qualitation and positioning study, as well as relationship with embryonic liver hematopoietic function during embryonic liver development. OBJECTIVE: To study the relationship between the E-selectin expression and the morphodifferentiation of the hepatic cells, the sinusoids endothelium as well as hematopoiesis during the embryonic development of mouse liver. METHODS: The mouse fetuses or fetal liver tissues from embryo day 11.5 (E11.5) to postnatal day 15.5 (P15.5) were dissected, fixed and embedded in paraffin. The sections were stained with hematoxylin and eosin and subjected to immunohistochemistry. The development of liver structure and the morphology of cells were observed under an optical microscope. The immunohistochemistry was taken to investigate the expression and localization of E-selectin in fetal livers at different developmental stages. RESULTS AND CONCLUSION: The progenitor liver cells gathered to form the hepatic parenchymal cords at E11.5. The hepatic parenchymal cords were separated by sinusoids with sporadic haemopoietic stem cells in it. The progenitor liver cells began to proliferate and differentiate at E12.5. From then on, the parenchymal cells increased, the volume of the hepatocytes became larger and the karyoplasmic ratio was decreased. The hepatic lobules were formed at natal time. The lacuna of the hepatic sinusoid became narrower and the endothelial cells grown to contiguous. At E12.5, the hematopoietic cells began its hematogenesis and reached its peak at E13.5-E15.5, and then both the blood-forming tissue and hematogenesis decreased gradually. E-Selectin expressed in the membrane of the endothelial cells from E11.5 to E15.5, located in endothelial cell membrane, and disappeared gradually along with the development of endothelial cells and the maturation of the hepatic cells. Above-mentioned results indicated that the most important time for the development of the individual cells in fetus liver is E12.5-E15.5. The expression of E-Selectin was appeared in the sinusoid endothelium, which is associated with the haematogenesis of fetus liver and the differentiation of hepatic cells. Related Articles | Metrics

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Adeno-associated virus vector-medicated green fluorescent protein transfected human amniotic epithelial cells in vitro   Luo Hong-wu, Huang Xiang-jun, Liu Xun-yang, Huang Fei-zhou 2010, 14 (10):  1843-1846.  doi: 10.3969/j.issn.1673-8225.2010.10.028 Abstract ( 399 )   PDF (750KB) ( 514 )   Save BACKGROUND: Human amniotic epithelial cells (AECs) are easy to obtain and can function as ideal seed cells for cell transplantation and tissue repair. Currently, marking and tracing of human AECs remains rarely reported. OBJECTIVE: To explore the efficiency of adeno-associated virus (AAV) vector-medicated green fluorescent protein (GFP) on in vitro cultured human AECs transfection. METHODS: Human amnion samples were harvested and trypsinized to isolate human AECs. The AECs were transfected with AAV-GFP, and the transfection efficiency was detected. RESULTS AND CONCLUSION: Human AECs were successfully primary cultured and passaged in vitro. AAV-GFP-transfected AECs stably and highly expressed GFP, with a transfection efficiency of 58%. Related Articles | Metrics

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Non-virus vector methods in hBDNF gene transfected bone marrow mesenchymal stem cells: Lipofectamine versus electroporation Chen Guan-gui, Liu Qian-xu, Xie Ding-hua 2010, 14 (10):  1847-1852.  doi: 10.3969/j.issn.1673-8225.2010.10.029 Abstract ( 373 )   PDF (600KB) ( 516 )   Save BACKGROUND: Gene transfection of cells includes virus and non-virus vector. As virus vector has some issues, such as safety and immunological rejection, the present study explored lipofectamine and electroporation transfection methods.    OBJECTIVE: To establish genetic engineering cells using human brain-derived neurotrophic factor (hBDNF) gene transfected bone marrow mesenchymal stem cells (BMMSCs) by lipofectamine or electroporation, and explore its characteristics and expression in vitro. METHODS: Lipofectamine method: The BMMSCs were obtained from the tibias and femurs of the guinea pigs. The third passage BMMSCs were cultured with plasmid-lipofectamine mixture for 6 hours, followed by fetal bovine medium for 48 hours. Immunohistochemistry was performed for transient expression. G418 was added after 48 hours. Electroporation method: BMMSCs were trypsinized and resuspended with serum-free medium. Cell suspension was added into electrotransformation pool, and plasmid was added. The electrotransformation pool was moved between electrodes. After transfection for 48 hours, gene transient expression was detected. G418 was added after 48 hours. Brain-derived neurotrophic factor expression was detected by immunohistochemistry and RT-PCR. RESULTS AND CONCLUSION: Immunohistochemistry showed that BDNF transient expression was 5.80% by lipofectamine and 24.29% by electroporation. Cells almost died at 14 days following lipofectamine transfection. Stable expression cell lines of BDNF engineered BMMSC were successfully established by electroporation, with 90% expressive rate by immunohistochemistry and expression of BDNF mRNA by RT-PCR. Genetic engineering cells using BDNF transected BMMSC were established by electroporation whereas failed by lipofectamine, and the expressed BDNF was confirmed by immunohistochemistry and RT-PCR in vitro. Related Articles | Metrics

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Mesencephalic neural stem cell transgene for treating Parkinson’s disease: Possibility and feasibility? Ding Ji-gu 2010, 14 (10):  1855-1860.  doi: 10.3969/j.issn.1673-8225.2010.10.031 Abstract ( 316 )   PDF (531KB) ( 590 )   Save BACKGROUND: Mesencephalon-derived neural stem cells (NSCs) differentiate into dopaminergic neurons in hypoxia and transgenic condition with body culture morphology and function of adequate maturity of dopamine neurons. Mesencephalon-derived NSCs become ideal seed cells for the treatment of Parkinson's disease with the rapid development of stem cell transplantation in the past few years. OBJECTIVE: To review research progress of mesencephalon-derived NSCs transgene for treating Parkinson's disease. METHODS: PubMed database was retrieved in computer (1992-01/2006-12), with the key words of “Neural Stem Cell(NSC), Mesencephalic Neural Stem Cell (M-NSC), Neural Stem Cell Transplant, Parkinson’s Disease (PD)”. Simultaneously, China Journal Full-Text Database (2003-01/2008-12) was retrieved with the same key words. RESULTS AND CONCLUSION: The NSCs and in the basic research of M-NSCs and M-NSCs transplantation in the treatment of PD-related documents were selected. A total of 83 documents were collected. Following reading titles and abstracts, 14 studies with irrelative objectives and contents, 16 studies with repetitive contents and 53 articles of Meta analysis were excluded. Totally 47 literatures were included. The main pathologic changes of Parkinson's disease are a dopaminergic neuron degeneration of necrosis in the brain substantia nigra. Using genetically modified neural stem cells in treatment of Parkinson's disease is the most recognized as the most promising treatment. In the hypoxia conditions, transgenic culture of brain-derived neural stem cells can be effectively induced to differentiate into dopaminergic neurons, provide experimental basis for in vitro sufficient amount of dopamine neuron transplantation in the treatment of PD. Basic research of neural stem cells have achieved certain results, but clinical and experimental study of neural stem cell transplantation in the treatment of PD is still stuck in the laboratory stage. Mesencephalic neural stem cell biological characteristics, isolation, proliferation and differentiation are subject to further study. In addition, biological safety problems using mesencephalic neural stem cells in the human body gradually attracted the attention of the people. In vitro neural stem cells of many passages would occur, and will bring certain side effects, and tumorigenicity remains in further exploration. Related Articles | Metrics

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Research and progress of cancer stem cells and tumor angiogenesis Zhang Hua-feng, Sheng Yu-wen, Jiang Hua-mao 2010, 14 (10):  1861-1864.  doi: 10.3969/j.issn.1673-8225.2010.10.032 Abstract ( 311 )   PDF (408KB) ( 463 )   Save BACKGROUND: Recent investigations show that there are a small part of self-renewing and multi-potent cells named as tumor stem cells. Those cells share many characteristics with somatic and embryonic stem cells and are thought to be responsible for driving tumor progression in growing list of neoplastic diseases. Vascular endothelial growth factor (VEGF) is an important angiogenesis factor, which can regulate endothelial proliferation, angiogenesis, permeability of blood vessel, and thrombogenesis. OBJECTIVE: To summarize the progress of cancer stem cells and tumor angiogenesis. METHODS: A computer-based online search was conducted in PUMMED, CNKI, and Wanfang databases with the key words of “cancer stem cells, vascular endothelial growth factor, new vascularize” in both Chinese and English from January 2000 to October 2009. RESULTS AND CONCLUSION: Among 153 articles, there were 13 in Chinese and 140 in English. Titles and abstracts were preliminarily screened, and articles which were non-relative (n=35), duplicated (n =30), and Meta analysis (n =57), were excluded. A total of 31 articles were included in the final analysis. Angiogenesis regulated by multiple factors was a necessary link for tumor growth and transferring. Stem cells promoted angiogenesis, thereby promoted tumor growth and transferring. However, the modified stem cells caused the opposite effects. On the other hand, the stem cells were considered as a substance vector to localize tumor focus, in particular, it will be potential for looking for subclinical focus and distal focus. Related Articles | Metrics

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Application of hepatic stem cell transplantation to liver disease treatment Xu Gui-juan, Jia Lian-qun, Wu Yun-hai, Yan Ying-chun, Chen Yang 2010, 14 (10):  1865-1868.  doi: 10.3969/j.issn.1673-8225.2010.10.033 Abstract ( 466 )   PDF (413KB) ( 414 )   Save BACKGROUND: At present, the problems such as serious shortage of donor liver organs for transplantation, surgical injury, high incidence of surgical complications, as well as the high costs limit the development of liver transplantation, while the hepatic stem cell (HSC) transplantation provides a new pathway for the treatment of end-stage liver disease. OBJECTIVE: To introduce the source and classification of HSCs, research progress and problems of HSC transplantation for treatment of end-stage liver disease, and the clinical application prospects of HSC transplantation. METHODS: Articles were collected from CNKI and Medline database with the keywords of “hepatic stem cells, liver disease, transplantation” in both Chinese and English from 1999 to 2009. Among 87 articles, 30 were included according to inclusion and exclusion criteria. Following reading titles and abstracts, original articles, and articles closely related to HSC transplantation with reliable argument and evidence and general analysis were included. Articles of repetitive studies and poor quality were excluded. RESULTS AND CONCLUSION: The HSC can be divided into liver-derived stem cells and non-liver-derived stem cells. Liver-derived stem cells include hepatic oval cells, mature liver cells and small hepatocyte-like progenitor cell. Non-liver-derived stem cells were mainly derived from embryonic stem cells, bone marrow hematopoietic stem cells and pancreatic stem cells. Currently, the research for the treatment of liver disease by HSC is still in its early stages. There are many difficult issues to be studied and solved in the discovery, separation, purification, comprehensive identification, cultivation, directed differentiation as well as clinical trials. However, as a new source of seed cells, HSC can not only replace the damaged tissue but can stimulate the receptor in tissue regeneration. Hence, compared with the clinical liver transplantation and bio-artificial liver, there are very bright future for the treatment of liver diseases by transplating HSC. Related Articles | Metrics

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Isolation methods and biological characteristics of bone marrow mesenchymal stem cells Li Lu-sheng, Zhang Han, Wang Cheng-jun, Cheng Hong-bin, An Yi-hua 2010, 14 (10):  1869-1873.  doi: 10.3969/j.issn.1673-8225.2010.10.034 Abstract ( 327 )   PDF (493KB) ( 630 )   Save BACKGROUND: Bone marrow mesenchymal stem cells (BMSCs) have the properties involving high proliferation capability, widely distribution, functional tissue repair after injury, as well as immune modulation, by which bring us extensive therapeutic possibilities. There are plenty of methods for isolation of BMSCs, yet, BMSCs exhibit discrepancies in varied growth stage and culture conditions. Up to now, there has been no agreement about the identification methods for cultured BMSCs. OBJECTIVE: To review the isolation methods and biological characteristics of BMSCs, and to compare the differential expression of BMSCs between in serum and serum-free medium, prior to and after proliferation, as well as before and after induction. METHODS: A computer-based online search was performed using key words of “bone marrow mesenchymal stem cells, isolation, culture, induce, marker, and characterization” to find documents published in the database of CNKI (http://dlib.cnki.net/kns50/) or Pubmed (http://www.ncbi.nlm.nih.gov/PubMed) from January 2003 to June 2009. The languages were limited Chinese and English. A total of 237 literatures were searched by the computer.  RESULTS AND CONCLUSION: The positive rates of CD44 and CD34 of BMSCs isolated by the whole bone marrow culture were smaller than that of the density gradient centrifugation. However, BMSCs isolated by the whole bone marrow culture were superior to those isolated by the density gradient centrifugation in cell viability, proliferation rate, confluence time, as well as generation time. Other methods for BMSCs isolation had drawbacks of large cost and high requirement of experimental equipments. Following conditions were used to identify BMSCs: cell adherence, cell surface molecule labeling, strong self-proliferation ability, as well as potentials multi-directional differentiation. BMSCs exhibit differential expression between in serum and serum-free medium, prior to and after proliferation, as well as before and after induction. Related Articles | Metrics

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Umbilical cord blood and blood plasma infusion for treating hepatitis: Grouping control and 1-year follow-up Chen Li-min, Tang Xiao-peng, Gong Huan-yu 2010, 14 (10):  1874-1877.  doi: 10.3969/j.issn.1673-8225.2010.10.035 Abstract ( 281 )   PDF (368KB) ( 490 )   Save BACKGROUND: Umbilical cord blood transplantation can ameliorate hepatic and immunologic function, repair hepatic injury, and promote hepatic regeneration, however, the differentiation mechanism and biological characteristics remain poorly understood, and the long-term efficiency need to be explored.   OBJECTIVE: To investigate the long-term therapeutic efficacy of infusing umbilical cord blood and blood plasma in treating chronic severe hepatitis B patients. METHODS: Totally 50 chronic severe hepatitis B patients received treatment at the Second Xiangya Hospital, Central South University from January 2003 to January 2004 were randomly divided into the treatment and control groups, with 25 cases in each group. All patient were accepted an ordinary synthetic treatment, and the differences between age, pathogenetic condition, medication had no significance. The umbilical cord blood was obtained from healthy full-term spontaneous delivery parturient, centrifugated, remained karyotes and cord plasma, and used within 24 hours. Patients in the treatment group were received umbilical cord blood infusion, 200 mL once, 1-2 times per week, totally, each patients infused 4-8 times (mean 5 times); those in the control group were infused with adult fresh blood plasma. The changes of hemogram and hepatic function were measured.  RESULTS AND CONCLUSION: All the patients were followed-up for 1 year. The hemogram and hepatic function indexes were similar in the 2 groups before treatment (P > 0.05). The hemogram index had no obviously difference at 1 year after treatment (P > 0.05), but the alanine aminotransferase and total bilirubin levels were decreased in the treatment compared with the control group (P < 0.05), but the albumin was significantly increased (P < 0.05). Compared with before treatment, the platelet level had no significant changes at 1 year after treatment, but the alanine aminotransferase and total bilirubin levels were deeply decreased (P < 0.05), albumin was significantly increased (P < 0.05); the platelet and albumin levels were dramatically decreased in the control group (P < 0.05). It suggested that umbilical cord blood infusion can improve the hepatic function and hemogram; therefore, it can be served as supplementary therapeutic measure for severe hepatitis. Related Articles | Metrics

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Tear film stability after pterygium excision and autologous corneal limbal stem cells transplantation versus simple pterygium excision Huang Jiang, Xu Guo-xu, Wei Xiao-hong, Bu Shu-yang, Tang Hua, Zhang Ji, Ji Xiao-yan 2010, 14 (10):  1878-1881.  doi: 10.3969/j.issn.1673-8225.2010.10.036 Abstract ( 258 )   PDF (313KB) ( 487 )   Save BACKGROUND: Treatment methods of pterygium mainly include pterygium excision, pterygium excision combined with local application of mitomycin, pterygium excision combined with conjunctival flap transfusion and pterygium excision combined with autologous corneal limbus stem cell transplantation. Dry eye commonly occurred in many patients following pterygium excision. OBJECTIVE: To evaluate the effects of stem cell transplantation of limbus cornea and simple excision of pterygium on tear film stability. METHODS: Eighty patients (eighty eyes) with pterygium were involved in this clinical experiment. All patients were randomly divided into 2 groups: Group A accepted simple excision of pterygium in 40 patients with 40 eyes under a 10-fold microscope, and Group B accepted excision of pterygium with stem cell transplantation of limbus cornea in 40 patients with 40 eyes under a 10-fold microscope, in which a free transplantation of the superotemporal limbus stem with an adjacent piece of conjunctiva was transplanted in the excision area. Slit-lamp examination, tear film break-up time and questionnaire on dry eye were performed before operation, at one week post-operation, and at three months post-operation. RESULTS AND CONCLUSION: In both groups, following surgery, some patients affected dryness, foreign body sensation, burning sensation. These symptoms were more in the group A compared with group B (P < 0.05). Implant was red 1 week following surgery in the group B, and confluence was found, without infection or rejection. The tear film break-up time was prolonged in the group B compared with the group A at 1 week following surgery, and no significant difference was determined at 3 months. Results indicated that compared with simple excision of pterygium, combined excision of pterygium with stem cell transplantation of limbus cornea obtained better outcomes, and could decrease the manifestations of dry eye and maintain better tear film stability in patients with pterygium. Related Articles | Metrics

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Autologous peripheral blood hemopoietic stem cell transplantation in combination with bortezomib and high-dose melphalan for multiple myeloma in 3 cases Sun Zhi-qiang, Wang Ji-shi, Lu Ying-hao, Xie Run-lan, Long Zheng-mei 2010, 14 (10):  1882-1884.  doi: 10.3969/j.issn.1673-8225.2010.10.037 Abstract ( 296 )   PDF (336KB) ( 414 )   Save BACKGROUND: Autologous peripheral blood hemopoietic stem cell transplantation (HSCT) in combination with high-dose chemotherapy significantly improves complete remission and survival rate of multiple myeloma patients. However, the relapse rate is high. Bortezomib is 26S proteasomes inhibitor, and effective on the primary treatment of multiple myeloma. OBJECTIVE: To evaluate the curative effect of HSCT in combination with bortezomib and high dose-melphalan for multiple myeloma. METHODS: A retrospective analysis of 3 patients with a stage- Ⅲ multiple myeloma admitted to Department of Hematology, Affiliated Hospital of Guiyang Medical College from October 2006 to May 2007, was conducted. Chemotherapy and granulocyte colony-stimulating factor were used to mobilize autologous peripheral blood hemopoietic stem cells. All patients were pretreated with 200 mg/m2 melphalan via intravenous drip 3 days before transplantation, followed by HSCT 48 hours after drug termination. RESULTS AND CONCLUSION: All patients obtained prompt and sustained hematopoietic reconstitution, and bone marrow depression restored 30 days following HSCT. Case 1 and 2 obtained complete remission, and case 3 obtained partial remission. Results show that HSCT in combination with bortezomib and high-dose melphalan is a safe and feasible treatment on multiple myeloma. The patients have good tolerance to pretreatment. Related Articles | Metrics

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Bone marrow mesenchymal stem cells transplantation for the treatment of sclerodermatous chronic graft-versus-host disease: Immunologic mechanism changes in 4 cases Zhou Hong, Guo Mei, Sun Qi-yun, Huang Shan, Yang Zhuo, Bian Chun-jing, Zeng Yang, Ai Hui-sheng, Zhao Chun-hua 2010, 14 (10):  1885-1891.  doi: 10.3969/j.issn.1673-8225.2010.10.038 Abstract ( 280 )   PDF (593KB) ( 1091 )   Save BACKGROUND: The immunomodulatory ability of bone marrow mesenchymal stem cells (BMSCs) gives it a promising future in treating graft-versus-host disease (GVHD), especially with previous success in treating patients with acute GVHD. However, there are fewer reports concerning BMSCs in treating chronic GVHD, particularly for sclerodermatous chronic graft-versus-host disease (ScGVHD). OBJECTIVE: To evaluate the efficacy and safety of treatment of BMSCs for ScGVHD, and to primarily explore the immunological mechanism of clinical efficacy. METHODS: Four ScGVHD patients at the Affiliated Hospital of Academy of Military Medicine Science, between September 2006 and August 2008, were enrolled for this trial. The median patient age was 41 years, 1 female and 3 male. The patients received BMSCs infusion at a dose of (1.0-2.0)×107 cells every time by intrabone marrow injection from the anterosuperior iliac spine and BMSCs from the same donor for the same patient were infused more than once. Concomitant medications for ScGVHD were individualized for each patient, but all were current standard medicines and the doses were significantly tapered. RESULTS AND CONCLUTION: After BMSCs infusion, the ratio of Th1 to Th2 was dramatically overturned, with an increase of Th1 and a decrease of Th2 reaching at a new balance. Correspondingly, symptoms of all the four patients gradually improved. During the course of BMSCs treatment, the life signs and laboratory results from the recipients remained normal. By the time of this report, there has been no recurrence of leukemia in the four patients. Although this study alone cannot guarantee the application of BMSCs in ScGVHD, the results are strongly in favor of the idea that the BMSCs treatment for ScGVHD patients is therapeutically practical without any detectable side effects, which may provide a new insight into the matter of treating ScGVHD clinically, thus will greatly increase the survival rate of leukemia after allogeneic bone marrow transplantation. Related Articles | Metrics

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Schwann cells purification by four different methods in vitro Chen Gang, Yang Cai-hong, Tian Lin-qiang, Guo Feng-jin, Chen An-min, Sun Kai 2010, 14 (10):  1892-1896.  doi: 10.3969/j.issn.1673-8225.2010.10.039 Abstract ( 287 )   PDF (336KB) ( 420 )   Save BACKGROUND: Schwann cell is one of the major seed cells in peripheral nervous system and plays an important role in neural injury and neural disease. However, the source of Schwann cells is limited. And the purity of Schwann cells is affected due to the pollution of fibroblasts. Many purified methods have been proposed, but every one has its defect to satisfy the clinical demand. OBJECTIVE: To compare the differences among differential adhesion purified method, cold jet purified method, immunomagnetic beads selection purified method and G418 selection purified method to purify Schwann cells of neonatal rat in vitro. METHODS: Bilateral sciatic nerves of SD rats were harvested under sterile condition. Schwann cells were purified respectively using differential adhesion purified method, cold jet purified method, immunomagnetic beads selection purified method and G418 selection purified method. Cell viability was compared, and cell purity was determined by immunohistochemistry.  RESULTS AND CONCLUSION: The purity of Schwann cells separated by differential adhesion method was low, but the viability was fair. The purity and viability of cells following cold jet method immunomagnetic beads selection method was high. The purity of cells separated by immunomagnetic beads selection methods was similar to that of cold jet method immunomagnetic beads selection method, but the cell viability was worse. The cell viability following G418 selection method was bad, but the purity was high. Related Articles | Metrics

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Differentiation potential of monocytes into lymphatic endothelial cells Liang Yan-hong, Zhang Zhao-lin, Tian Hua, Wang Chang-ming, Wang Shi-kun, Li Xin, Song Tao 2010, 14 (10):  1897-1900.  doi: 10.3969/j.issn.1673-8225.2010.10.040 Abstract ( 270 )   PDF (320KB) ( 434 )   Save BACKGROUND: Previous studies have shown that monocytes can transdifferentiate into vascular endothelial cells under the induction of various factors including vascular endothelial growth factor (VEGF). It remains poorly understood whether monocytes can be induced to transdifferentiate into lymphatic endothelial cells in vitro. OBJECTIVE: To explore the possibility of the transdifferentiation of monocytes into lymphatic endothelial cells under inflammatory condition. METHODS: Fresh monocytes from peripheral blood were collected by Ficoll density gradient centrifugation and cultured in an endothelial cell medium, followed by incubation in fibronectin-plated well or treated with tumor necrosis factor α for 24 hours, respectively. The expression of specific markers of lymphatic endothelial cells, such as LYVE-1, Podoplanin, Porx-1 and VEGF receptor 3 (VEGFR-3), as well as the endothelial cells markers, such as vWF, endothelial nitric oxide synthase (eNOS) and VEGFR-2, were detected by RT-PCR and immunochemical methods. RESULTS AND CONCLUSION: Prior to induction, monocytes were positive to LYVE-1, but negative for Podoplanin, Porx-1, and VEGFR-3, vWF, eNOS, as well as VEGFR-2. Following induction, the cultured mononcytes were positive for Podoplanin, Prox-1 and VEGFR-3, but remained negative for vWF, eNOS and VEGFR-2. It suggested that monocytes can be induced to express the markers of lymphatic endothelial cells stimulated by fibronectin or tumor necrosis factor α. Related Articles | Metrics