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    03 December 2015, Volume 19 Issue 50 Previous Issue    Next Issue
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    Bone marrow mesenchymal stem cells from multiple myeloma patients aberrantly affect chemotactic function of myeloma cell lines
    Fei Xiao-ming, Li Jun-xia, Tang Yu, Lei Fang, Lu Hua
    2015, 19 (50):  8037-8042.  doi: 10.3969/j.issn.2095-4344.2015.50.001
    Abstract ( 244 )   PDF (527KB) ( 504 )   Save

    BACKGROUND: Although bone marrow mesenchymal stem cells from multiple myeloma patients present a variety of abnormalities, it is unclear how these abnormal mesenchymal stem cells influence the chemotactic function of myeloma cell lines.
    OBJECTIVE: To investigate the in vitro effects of bone marrow mesenchymal stem cells from normal donors versus multiple myeloma patients on the chemotactic capacity of myeloma cell lines.
    METHODS: In vitro cultured bone marrow mesenchymal stem cells derived from either normal donors (N-MSCs group) or newly-diagnosed multiple myeloma patients (MN-MSCs group) were directly co-cultured with U266 cells, in the presence or absence of bortezomib; and then harvested U266 cells were assayed for Transwell migration 
    and mRAN expression of chemotaxis-related genes. U266 Transwell migration to conditioned medium derived from either N-MSCs or MN-MSCs was also tested.
    RESULTS AND CONCLUSION: After co-cultured with N-MSCs or MN-MSCs, U266 cells harvested from MN-MSCs group showed increased spontaneous Transwell migration and up-regulated CCR1 mRNA level than those from N-MSCs group (P < 0.05), whatever bortezomib was present or not. However, there was no evident difference between U266 cell Transwell migration to conditioned medium derived from either MM-MSCs group or N-MSCs group. Our study implies that there may be some intrinsic aberrance in bone marrow mesenchymal stem cells derived from multiple myeloma patients, which can modulate the chemotactic migration of myeloma cell lines when they directly interact with each other.
     

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    Primary culture and biological characteristics of bone marrow mesenchymal stem cells from a rabbit
    2015, 19 (50):  8043-8047.  doi: 10.3969/j.issn.2095-4344.2015.50.002
    Abstract ( 517 )   PDF (478KB) ( 628 )   Save

    BACKGROUND: Rabbit bone marrow mesenchymal stem cells as a kind of adult stem cells with strong proliferation and multilineage differentiation potential exhibit a tremendous application potential in tissue engineering and biological therapy.
    OBJECTIVE: To in vitro culture, proliferate and identify rabbit bone marrow mesenchymal stem cells and to observe cell biological characteristics.
    MEHTODS: Bone marrow of rabbits was extracted under sterile conditions to separate bone marrow mesenchymal stem cells using the whole bone marrow adherence method and Percoll density gradient centrifugation method. Afterwards, the cells were purified and proliferated using differential adherence method. Morphology and growth pattern of cells were observed under microscope, and expression of cell surface antigen markers was detected by flow cytometry.
    RESULTS AND CONCLUSION: Rabbit bone marrow mesenchymal stem cells presented with short adherent time and fast growth. After passage and purification, impurities cell counts were decreased. Primary cells presented with triangular, fusiform and spindly shapes. Passage 5 cells with single shape showed the typical polar swirling growth, and could not express CD34 and CD45, but expressed CD29 and CD44. These findings indicate that the cells cultured using the whole bone marrow adherence method and Percoll density gradient 
    centrifugation method possess stem cell characteristics in morphology, surface markers and multilineage differentiation, which have been identified as bone marrow mesenchymal stem cells by flow cytometry.  

     

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    let-7f effects on the proliferation of bone marrow mesenchymal stem cells
    2015, 19 (50):  8048-8055.  doi: 10.3969/j.issn.2095-4344.2015.50.003
    Abstract ( 243 )   PDF (704KB) ( 474 )   Save

    BACKGROUND: There is no clear understanding about the effect of let-7f and interleukin-6 (IL-6) on the proliferation of bone marrow mesenchymal stem cells and their relationship.
    OBJECTIVE: To explore the effects of expression levels of let-7f and IL-6 on the proliferation of bone 
    marrow mesenchymal stem cells and their relationship.
    METHODS: (1) LV-rno-let-7f-up and LV-rno-let-7f-down were constructed and transfected into bone marrow mesenchymal stem cells of Sprague-Dawley rats, respectively. Then, there were four groups in the study: transfection upregulation group transfected with LV-rno-let-7f-up), transfection inhibition group (transfected with LV-rno-let-7f-down), negative control group (transfected with FU-RNAi-NC-LV), and untransfected group. The expression level of let-7f in each group was detected by qRT-PCR. The proliferation ability of cells and expression levels of IL-6 when let-7f expression was at different levels were detected by MTT, flow cytometry and ELISA. The expression of Cyclin D1 at mRNA and protein levels was detected by qRT-PCR and western blot, respectively. (2) To predict the potential target gene of let-7f, the wild-type/mutant IL-6 3’UTR reporter gene vectors were constructed, and cotransfected with let-7f/let-7f inhibitor respectively into the 293T cells to measure the luciferase.
    RESULTS AND CONCLUSION: Compared with the negative control group, the proliferative and cloning capacities of cells in the transfection upregulation group were higher; the number of cells was significantly decreased at G1 stage and increased at S stage, and the apoptotic cells were reduced in number (P < 0.05). However, the transfection inhibition group had opposite results. The expression level of IL-6 in the transfection upregulation group was lower than that in the untransfected group and negative control group (P < 0.05); while in the transfection inhibition group, the expression level of IL-6 was significantly increased (P < 0.05). The expression of Cyclin D1 at mRNA and protein levels was up-regulated in transfection upregulation group (P < 0.05) and down-regulated in the transfection inhibition group (P < 0.05), but there was no significant difference between the negative control group and untransfected group (P > 0.05). Luciferase activity of cells transfected with wide-type IL-6 3’UTR and let-7f was significantly reduced (P < 0.05). These findings indicate that up-regulation of let-7f can promote the proliferative and cloning capacities of bone marrow mesenchymal stem cells and reduce cell apoptosis, but downrelation of let-7f exhibits an inhibitory effect. Overexpression of IL-6 can suppress the proliferation of bone marrow mesenchymal stem cells, which is considered to be a target gene of let-7f, and let-7f may suppress the expression of IL-6 to promote the cell proliferation. 

     

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    Down-regulation of miR-221-3p/222-3p inhibits cell proliferation and promotes chondrogenic differentiation of human bone marrow mesenchymal stem cells
    Yan Ji-hong, Yang Shu, Sun Hai-mei, Cao Dan-dan, Zhang Xiu-ying, Ji Feng-qing, Guo Duo, Wu Bo,
    2015, 19 (50):  8056-8061.  doi: 10.3969/j.issn.2095-4344.2015.50.004
    Abstract ( 283 )   PDF (533KB) ( 771 )   Save

    BACKGROUND: The use of mesenchymal stem cells in the field of tissue engineering for osteoarticular injury repair is a very promising tool since these cells are readily expandable and able to differentiate into chondrocytes. Abundant evidence suggests that microRNAs play critical roles in chondrogenic differentiation of mesenchymal stem cells.
    OBJECTIVE: To observe the chondrogenic effect of human bone marrow mesenchymal stem cells transfected with lentiviral vectors bearing miR-221-3p/222-3p inhibition, thereby provding new strategies for cartilage injury.
    METHODS: miRNA microarray technology was applied to detect microRNAs expression profiles at three different stages of chondrogenic differentiation induction after transforming growth factor-β3 treatment and verified by 
    real-time fluorescence quantitative PCR (RT-qPCR). Human bone marrow mesenchymal stem cells were infected with lentivirus bearing miR-221-3p/222-3p inhibition. After co-suppressing the expression of miR-221/222-3p, cell counting kit-8 was used to determine the cell proliferation, the differentiation of bone marrow mesenchymal stem cells towards chondrocytes was verified by type II collagen protein expression through immunohistochemistry and glycosaminoglycan accumulation was also elevated by sarranine O staining. RT-PCR was used to detect type II collagen and aggrecan mRNA expression at 21 days of chondrogenic induction.
    RESULTS AND CONCLUSION: The expression of miR-221-3p/222-3p was inhibited after Lv-miR221-3p/222-3p inhibition co-transfected into bone marrow mesenchymal stem cells. microRNA microarray and RT-qPCR results showed that the expression of miR-221-3p/222-3p was declined significantly at the anaphase of chondrogenic differentiation. The expression levels of chondrogenic markers, Aggrecan and type II collagen were significantly increased in the miR-221-3p/222-3p inhibition group and cell proliferation was also inhibited significantly compared with non-transduced cells or transduced with the empty lentiviral vector group. miR-221-3p/222-3p knockdown in bone marrow mesenchymal stem cells could inhibit proliferation but promote chondrogenic differentiation of bone marrow mesenchymal stem cells.

    中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

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    Breast cancer stem cells: separation and resistance protein analysis
    Guo Meng, Wan Li-xin
    2015, 19 (50):  8062-8066.  doi: 10.3969/j.issn.2095-4344.2015.50.005
    Abstract ( 258 )   PDF (450KB) ( 420 )   Save

    BACKGROUND: Breast cancer stem cells are relatively special cells in the body, which have the self-renewal and multi-differentiation ability to promote tumor formation and development, and maintain tumor growth for a long-term. Therefore, it is of great significance to analyze the expression of resistance proteins of breast cancer stem cells.
    OBJECTIVE: To isolate breast cancer stem cells from human breast cancer tissues, to observe their differentiation and morphology characteristics and to analyze their resistance proteins.
    METHODS: Thirty tumor samples of breast invasive ductal carcinoma were selected to separate single cell suspension using mechanical separation method, and breast cancer stem cells and differentiated cells were sorted with two-step immunomagnetic bead method. Two-step immunocytochemistry method was used to detect the expression of resistance proteins in breast cancer stem cells.
    RESULTS AND CONCLUSION: Percentage of breast cancer stem cells had no significant correlations with age, long diameter of the tumor, lymph node metastasis and histological grading (P > 0.05). P-gp and GST-π positive rates in the breast cancer stem cells were significantly higher than those in the differentiated cells (P < 0.05); while TopoII and LRP positive rates in the breast cancer stem cells were significantly lower than those in the differentiated cells (P < 0.05). To conclude, breast cancer stem cells show stronger drug resistance than the differentiated cells by highly expressing P-gp and GST-π and lowly expressing TopoII and LRP, which may be the key reason for chemotherapy resistance in breast cancer. 

     

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    Effect of different tumescent local anesthesia on the proliferation and differentiation of human adipose stem cells cultured in vitro
    王志刚,吴建萍,赵文香
    2015, 19 (50):  8067-8071.  doi: 10.3969/j.issn.2095-4344.2015.50.006
    Abstract ( 244 )   PDF (495KB) ( 407 )   Save

    BACKGROUND: Different local anesthetic solutions for tumescent liposuction are necessary and have a certain effect on the biological characteristics of human adipose stem cells.
    OBJECTIVE: To explore the effect of different tumescent local anesthesia on the proliferation and differentiation of human adipose stem cells cultured in vitro.
    METHODS: Passage 3 human adipose stem cells were intervened by different tumescent local anesthesia (bupivacaine, ropivacaine, lidocaine). Cells cultured in low-glucose DMEM served as controls. Proliferation ability, lactate dehydrogenase activity in the supernatant and percentage of cells under adipogenic differentiation were detected.
    RESULTS AND CONCLUSION: After intervention for 1, 2, 3, 4, 5 days, the supernatant lactate dehydrogenase activity in the bupivacaine, ropivacaine, lidocaine groups was significantly higher than that in the control group (P < 0.05), and moreover, the lactate dehydrogenase activity in the bupivacaine group was significantly higher than that in the ropivacaine group and lidocaine group (P < 0.05). The absorbance value of human adipose stem cells was lower in the bupivacaine, ropivacaine, lidocaine groups than the control group (P < 0.05) as well as lower in the bupivacaine group than the ropivacaine and lidocaine groups (P < 0.05). There was no significant difference in the percentage of cells under adipogenic differentiation between groups (P > 0.05). Overall, these findings indicate that different tumescent local anesthesia (bupivacaine, ropivacaine, lidocaine) can all inhibit the 
    proliferation of human adipose stem cells, but have no certain effects on the adipogenic differentiation.
     

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    Osteogenic differentiation of adipose-derived stem cells and the effect of melatonin on the bio-viability of differentiated cells
    Lu Tan, Wei Na, Zhang Chao, Dong Yu-zhen
    2015, 19 (50):  8072-8076.  doi: 10.3969/j.issn.2095-4344.2015.50.007
    Abstract ( 296 )   PDF (549KB) ( 567 )   Save

    BACKGROUND: Studies have indicated that melatonin can promote the differentiation of adipose-derived stem cells into neurons, and the effect of melatonin on the osteoblasts form adipose-derived stem cells is rarely reported.
    OBJECTIVE: To observe the osteogenic differentiation of adipose-derived stem cells and the effect of melatonin on the bio-viability of differentiated cells.
    METHODS: (1) Adipose-derived stem cells were isolated and purified from the inguinal fat of Kunming mice by type I collagenase digestion and differential adhesion method, respectively. Immunohistochemical staining of CD44 was used as a quality control. (2) Osteogenic induction medium was added to induce osteogenic 
    differentiation of passage 2 adipose-derived stem cells. Alkaline phosphatase staining and von Kossa method were combined to evaluate differentiation condition. (3) Melatonin at variable concentrations was added to treat mature osteocytes originated from adipose-derived stem cells and MTT was applied to determine their viability at 24 and 48 hours after culture respectively to find out optimal condition of melatonin treatment. (4) Melatonin at the optimal concentration was used to treat differentiated cells and detect alkaline phosphatase activity after 3 days and 6 days respectively.
    RESULTS AND CONCLUSION: (1) After seeding for 48 hours, most cells were adherent, and after 4 days, the cells displayed multiple shapes and colonies of different sizes formed. After subculture, cell morphology homogenized as spindle shape. Cells positive for CD44 were brownish yellow, and localized mainly on the cell membrane. (2) Differentiated cells were positive for von Kossa staining and black sediments scattered in the extracellular matrix. Alkaline phosphatase expressed positively, and brown-black particles, appeared within cells. (3) Melatonin supplement improved the viability of differentiated cells; and 1, 10 and 100 μmol/L was observed as the optimal concentrations both at 24 and 48 hours. (4) The intracellular alkaline phosphatatse activity was increased with time in all the groups (P < 0.05). Compared with the blank group, the intracellular alkaline phosphatase activity in Melatonin groups (1, 10 and
    100 μmol/L) had no changes at 3 days, but significantly increased at 6 days (P < 0.05). These findings indicate that melatonin can enhance the proliferation of osteocytes differentiated from adipose-derived stem cells, and improve the activity of intracellular alkaline phosphatase. 

     

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    MicroRNA-17-92 gene promotes renewal and proliferation of gastric cancer stem cells
    Song Zi-zheng, Yang Hua, Shang Yan-hong, Liu Bin, Zhang Gang,Wang Hua, Zang Ai-min
    2015, 19 (50):  8077-8083.  doi: 10.3969/j.issn.2095-4344.2015.50.008
    Abstract ( 259 )   PDF (616KB) ( 766 )   Save

    BACKGROUND: Studies have shown that microRNAs (miRNAs) have moderating effect on the renewal and differentiation of cancer stem cells. However, there is no complete understanding on the effect of microRNA-17-92 gene on gastric cancer stem cell renewal and proliferation.
    OBJECTIVE: To explore the effect of miRNA-17-92 in promoting self-renewal and proliferation of gastric cancer stem cells.
    METHODS: (1) The gradually reduced miRNAs during gastric cancer stem cell self-renewal were investigated using miRNA array based on RNAs from differentiated and adherent cells. (2) The miRNA-17-92 was constructed and transfected to gastric cancer stem cells. (3) The effects of miRNA-17-92 on the self-renewal of gastric cancer stem cells were studied by tumor sphere assay in vitro. (4) The effects of miRNA-17-92 on the proliferation of 
    gastric cancer stem cells were investigated by MTT assay and colony formation assay.
    RESULTS AND CONCLUSION: (1) miR-19b/20a/92a expression gradually reduced in the adhesion and differentiation of gastric cancer stem cells. (2) The expression of lentivirus carrying miRNA-17-19 gene in MKN28 cells and CD44-/EpCAM- cells were significantly increased; transient transfection of pre-miR-19b/20a/92a increased the expression of CD44-/EpCAM- and MKN28 miRNA, transient transfection of pre-miR-19b/20a/92a antagonists reduced the expression of SGC7901 and CD44+/EpCAM+ miRNA; overexpression of lenti-miR-19b/20a/92a significantly increased the ability of gastric cells to form tumor spheres; chemotherapy drugs prolonged the survival time of lenti-miR-19b/20a/92a-infected cells; transient transfection of pre-miR-19b/20a/92a significantly increased the number of CD44+/EpCAM+ cells, but transfection of pre-miR-19b/20a/92a antagonist reduced the number of CD44+/EpCAM+ cells. (3) MTT proliferation assay showed that gastric cancer cell proliferation rate in miR-19b/20a/92a stably expressing group was faster than that in the control group. Transient transfection of miR-19b/20a/92a precursor accelerated the growth rate of gastric cancer cells, and transient transfection of its antagonist slowed down the growth rate of gastric cancer cells. Colony formation assay showed that transient transfection of miR-19b/20a/92a precursor significantly increased the colony formation number as compared with the control group; transient transfection of miR-19b/20a/92a antagonist reduced the colony formation as compared with the control group. These findings indicate that miR-19b/20a/92a gene presents with continuous deletion in gastric cancer stem cell differentiation process, and miRNA-17-92 gene can promote the renewal and proliferation of gastric cancer stem cells. 

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    Molybdenum effect on ECA-109 cell chemosensitization and p75NTR cell inhibition
    Cai Zhong-li,Ding Chun-ping, Ji Sheng-hui, Liu Hong-feng, Chen Fan-zhou
    2015, 19 (50):  8084-8089.  doi: 10.3969/j.issn.2095-4344.2015.50.009
    Abstract ( 325 )   PDF (532KB) ( 767 )   Save

    BACKGROUND: In esophageal cancer chemotherapy, inhibiting proliferation of tumor cells and tumor stem cells can be effectively improved by using appropriate sensitive agents.
    OBJECTIVE: To explore the effect of molybdenum on ECA-109 cell chemosensitivity and p75NTR cell inhibition in esophageal carcinoma cells.
    METHODS: ECA-109 cells at logarithmic phase were selected and randomly divided into blank control group, cisplatin group, molybdenum group and molybdenum+cisplatin group (combination group). Molybdenum and cisplatin at different concentrations were used in the three groups. MTT assay was used to detect ECA-109 cell proliferation and growth; flow cytometry was used to detect the proportion of P75NTR cells.
    RESULTS AND CONCLUSION: Cisplatin at different concentrations showed a certain inhibitory role in ECA-109 cells, which had an increasing killing effect on esophageal carcinoma stem cells at dose- and time-dependent manner. Molybdenum alone had no remarkable killing effects on inhibiting ECA-109 proliferation and esophageal carcinoma stem cells. Combination of molybdenum and cisplatin was found to have an enhanced effect to inhibit ECA-109 cells and to kill esophageal carcinoma stem cells in a dose- and time-dependent manner, which was significantly different from the cisplatin group and blank control group (P < 0.05). These findings indicate that 
    molybdenum can promote and enhance the inhibitory effect of cisplatin on ECA-109 and p75NTR cells, which can be used as a chemosensitizer.
     中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

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    CD133, a stem cell marker, in neuroblastoma
    Fan Jia-rong, Li Wan-fu, Liang Ting
    2015, 19 (50):  8090-8094.  doi: 10.3969/j.issn.2095-4344.2015.50.010
    Abstract ( 460 )   PDF (464KB) ( 740 )   Save

    BACKGROUND: Neuroblastoma is the most common solid tumor in infants and children. Targeting cancer stem cell therapy can be expected to cure cancer radically, but because of small number, anti-apoptotic and anti-chemotherapy capacity, cancer stem cells are not sensitive to the radiotherapy and chemotherapy.
    OBJECTIVE: To explore the expression of stem cell marker CD133 in neuroblastoma and its clinical significance.
    METHODS: Fifty-eight children with neuroblastoma admitted at Department of Pediatric Surgery, the First Affiliated Hospital of Xinjiang Medical University, China from June 2004 to February 2014 were enrolled, including 46 cases of neuroblastoma and 12 cases of ganglion neuroblastoma. Then, the expression level of CD133 was analyzed by immunohistochemistry method, and the relationship between pathological types, International Neuroblastoma Staging System stage, survival time after surgery and the expression level of CD133 was explored.
    RESULTS AND CONCLUSION: There were 22 cases (48%) of neuroblastoma positive for CD133, and 4 cases (33%) of ganglion neuroblastoma positive for CD133. CD133 mainly expressed in the tumor cell cytoplasm. The expression rates of CD133 in different clinical stages were significantly different (P=0.011), which were 21% for stages 1 and 2, 64% for stages 3 and 4, 36% for stage 4S. And the positive rates of CD133 between patients with good prognosis and patients with poor prognosis were significantly different (P=0.031), which were 29% and 57%, respectively. The life cycle of CD133-negative patients was significantly longer than that of CD133-positive infants 
    (P < 0.05). There were tightly connections between CD133 and the occurrence, development, and prognosis of neuroblastoma, and thus, CD133 is of great significance to assess the survival time after surgery and improve of the diagnosis and treatment of neuroblastoma.  

     

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    Parental haploidentical peripheral blood stem cell transplantation for treatment of children with refractory severe aplastic anemia 
    Wan Ding-ming, Chen Xiao-na, Cao Wei-jie, Xing Hai-zhou, He Hai-yan, Liu Fei, Chen Shi-yu,
    2015, 19 (50):  8095-8101.  doi: 10.3969/j.issn.2095-4344.2015.50.011
    Abstract ( 290 )   PDF (537KB) ( 806 )   Save

    BACKGROUND: For pediatric patients with aplastic anemia in China, it is difficult to find human leucocyte antigen-matched sibling donors that are mostly replaced by parental donors.
    OBJECTIVE: To retrospectively analyze the clinical efficacy and safety of parental haploidentical peripheral blood hematopoietic stem cell transplantation in children with relapsed and refractory severe aplastic anemia.
    METHODS: Seventeen children with relapsed and refractory severe aplastic anemia who had no matched sibling or unrelated donor and failed to respond to immunosuppressive therapy were subjected to parental haploidentical peripheral blood hematopoietic stem cell transplantation. A conditioning regimen of fludarabine+cyclophosphamide+rabbit anti-human thymocyte immunoglobulin antibody and the triple therapy of methotrexate, cyclosporine A and mycophenolate mofetil were applied to prevent graft-versus-host disease.
    RESULTS AND CONCLUSION: (1) Of the 17 children, 16 cases (94%) reached hematopoietic reconstitution, and the median time of neutrophils ≥ 0.5×109/L and platelets ≥ 20×109/L was 13 (11-15) days and 17 (12-28) 
    days, respectively. (2) Incidence of acute graft-versus-host disease was 47% (8 of 17 cases), including 29% (5/17) of grades I-II and 18% (3/17) of grades III-IV. Incidence of chronic graft-versus-host disease was 41% (7/17). (3) With a median follow-up duration of 268 (43-753) days, the overall survival rate was 70.6% (12/17). Five dead cases (29%) belonged to transplantation-related death, including one case of fungal skin infections, one case of graft-versus-host disease, three cases of severe lung infection. No relapse case was reported. These findings indicate that if there are no matched sibling or unrelated donors and the immunosuppression effect is poor, parental haploidentical peripheral blood hematopoietic stem cell transplantation is a safe and effective salvage treatment for children with relapsed and refractory severe aplastic anemia.
    中国组织工程研究杂志出版内容重点:干细胞;骨髓干细胞;造血干细胞;脂肪干细胞;肿瘤干细胞;胚胎干细胞;脐带脐血干细胞;干细胞诱导;干细胞分化;组织工程

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    Autologous peripheral blood mononuclear cell transplantation following cell mobilization increases CD4+CD25+ regulatory T cells in rats with thromboangiitis obliterans
    Zhao Feng1, Deng Mo2
    2015, 19 (50):  8102-8107.  doi: 10.3969/j.issn.2095-4344.2015.50.012
    Abstract ( 214 )   PDF (478KB) ( 357 )   Save

    BACKGROUND: Autologous peripheral blood stem cells have pluripotent characteristics, promote ischemic limb angiogenesis, improve and restore the blood supply to the affected limb.
    OBJECTIVE: To observe the therapeutic effect of autologous peripheral blood mononuclear cell transplantation in rats with thromboangiitis obliterans.
    METHODS: Twenty-four rats were randomized into control, model and granulocyte colony stimulating factor (G-CSF) groups. In the latter two groups, animal models of thromboangiitis obliterans were established by femoral arterial injection of sodium laurate. In the G-CSF group, rats were injected with recombinant human G-CSF for 5 successive days for peripheral blood mononuclear cell mobilization, and then underwent peripheral blood mononuclear cell transplantation at day 7. In the control and model groups, rats were given normal saline injection.
    RESULTS AND CONCLUSION: After modeling, foot dorsum temperature and the percentage of CD4+CD25+ regulatory T cells decreased significantly in the model and G-CSF groups, and the area of leg ulcers was enlarged notably in these two groups. In the G-CSF group, the number of white cells and percentage of CD34+ cells to mononuclear cells were increased at 6 days after mobilization; the foot dorsum temperature and the percentage of CD4+CD25+ regulatory T cells increased significantly at 1 month after peripheral blood mononuclear cell transplantation, which were close to the normal values. These findings indicate that autologous 
    peripheral blood mononuclear cell transplantation following cell mobilization increases the percentage of CD4+CD25+ regulatory T cells in rats with thromboangiitis obliterans, thus promoting relief of symptoms and ulcer healing. 

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    Ginkgo-damole injection combined with bone marrow mesenchymal stem cell transplantation improves neurologic recovery from cerebral infarction
    Yang Chao-yang
    2015, 19 (50):  8108-8113.  doi: 10.3969/j.issn.2095-4344.2015.50.013
    Abstract ( 243 )   PDF (530KB) ( 386 )   Save

    BACKGROUND: Reconstruction of damaged brain tissue through cell transplantation has become a new way to treat cerebral infarction. In recent years, bone marrow mesenchymal stem cells have become the new darling in cell transplantation therapy.
    OBJECTIVE: To investigate the effect of ginkgo-damole injection combined with bone marrow mesenchymal stem cell transplantation to improve the neurological function of acute cerebral infarction rats and its mechanism.
    METHODS: Animal models of middle cerebral artery occlusion were made in rats using suture method, and then 60 rat models were randomly divided into control group, cell transplantation group and combination group. The control group was given intravenous injection of PBS via the tail vein; the cell transplantation group was given intravenous injection of bone marrow mesenchymal stem cell suspension (2.5×109/L) via the tail vein; the combination group was given intravenous injection of bone marrow mesenchymal stem cell suspension (2.5×109/L) and ginkgo-damole injection (2 mL/kg, once a day, totally 5 days) via the tail vein. Modified 
    neurological severity scores were recorded at 1, 3 days and 1, 2 weeks after transplantation. At 2 weeks after transplantation, expressions of brain-derived neurotrophic factor and growth associated protein 43 in the brain were detected using RT-PCR; cell apoptosis detected using MTT assay; BrdU positive cells counted using immunohistochemistry method.
    RESULTS AND CONCLUSION: There were no differences in the modified neurologic severity scores among the three groups at 1, 3 days after transplantation (P > 0.05), but the modified neurological severity scores in the combination group were lower than those in the cell transplantation group and control group at 1, 2 weeks after transplantation (P < 0.05). The expressions of brain-derived neurotrophic factor and growth associated protein 43 in the brain were significantly higher in the combination group than the other two groups at 2 weeks after transplantation (P < 0.05); compared with the other two groups, the number of apoptotic cells was less but the number of BrdU positive cells was higher in the combination group (P < 0.05). These findings indicate that the combination of ginkgo-damole injection and bone marrow mesenchymal stem cell transplantation can increase the expressions of brain-derived neurotrophic factor and growth associated protein 43 in the brain, inhibit cell apoptosis and improve neurological function in rats with cerebral infarction. 
     

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    Fleabane combined with bone marrow mesenchymal stem cell transplantation for cerebral infarction
    Lu Yi-zhong, Li He-hua, Lu Yi-fan
    2015, 19 (50):  8114-8119.  doi: 10.3969/j.issn.2095-4344.2015.50.014
    Abstract ( 277 )   PDF (511KB) ( 377 )   Save

    BACKGROUND: Cell transplantation becomes a new approach for treatment of cerebral infarction. In recent years, bone marrow mesenchymal stem cells (BMSCs) have become an important kind of seed cells in cell transplantation.
    OBJECTIVE: To investigate the effect of fleabane injection combined with BMSC transplantation on S100B protein and superoxide dismutase expression in acute cerebral infarction rats.
    METHODS: Animal models of acute cerebral infarction were made in Sprague-Dawley rats using suture method. After successful modeling, 80 model rats were randomly divided into control group, fleabane group, BMSC group and combined group (fleabane combined with BMSC transplantation). Changes of serum S100B protein and serum superoxide dismutase levels were detected using enzyme-linked immunosorbent assay and xanthine oxidase method, respectively, before and after treatment. NIHSS neurological function scores were measured to observe neurological behavior changes in model rats. The infarct volume was measured by TTC staining.
    RESULTS AND CONCLUSION: At 36, 7, 14 days after treatment, S100B protein levels in the fleabane group and BMSC group were significantly lower than that in the control group, but higher than that in the combined group (P < 
    0.05); serum superoxide dismutase levels in the fleabane group and BMSC group were significantly higher than that in the control group, but lower than that in the combined group (P < 0.05). At 1, 2, 3 weeks after treatment, NIHSS neurological function scores were ranked as follows: combined group < fleabane group and BMSC group < control group, and there was a significant difference between groups (P < 0.05). At 2 weeks after treatment, the infarct volume in the fleabane group and BMSC group was higher than that in the combined group but lower than that in the control group (P < 0.05). These findings indicate that fleabane combined with BMSC transplantation can inhibit the expression of S100B protein in rats with acute cerebral infarction, and promote the activity of superoxide dismutase, thereby playing a neuroprotective role. 

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    Effect of buyang huanwu decoction combined with bone marrow mesenchymal stem cell transplantation on cerebral ischemia-reperfusion injury
    Zhang Yun-ke, Song Jun-ying, Zhang Zhen-qiang
    2015, 19 (50):  8120-8125.  doi: 10.3969/j.issn.2095-4344.2015.50.015
    Abstract ( 217 )   PDF (526KB) ( 545 )   Save

    BACKGROUND: When acute cerebral ischemia attacks, matrix metalloproteinases (MMPs) lead to the occurrence of cerebral edema through degrading the extracellular matrix, breaking the close connection between endothelial cells, increasing the permeability of capillaries, and destroying the blood brain barrier.
    OBJECTIVE: From the aspects of MMPs and extracellular matrix, to discuss the therapeutic effects of buyang huanwu decoction combined with bone marrow mesenchymal stem cell (BMSCs) transplantation on cerebral ischemia-reperfusion injury.
     
    METHODS: A total of 96 Sprague-Dawley rats were randomly divided into model group, tissue inhibitor of MMPs-1 (TIMP-1) group, TIMP-1+BMSCs group (BMSCs group) and buyang huanwu decoction+BMSCs+TIMP-1 group (combined group that was divided into four subgroups, 12-, 24-, 36-, 48-hour groups). Rat models of cerebral ischemia-reperfusion were constructed, and TIMP-1 and BMSCs were injected to the brain of rats by a microinjector in a stereotaxic apparatus. Rats in the combined group were given buyang huanwu decoction (10 mL/kg), and rats in the other groups were given the same volume of normal saline at 7 days before surgery. After 10 days of administration, serum samples and brain tissues were collected to determine MMP-2 and MMP-9 levels using ELISA and to detect MMP-9 activity using gelatinases spectrometry method.
    RESULTS AND CONCLUSION: Compared with the model group, the levels of MMP-2 and MMP-9 in the serum and MMP-9 activity in the brain were decreased in the other groups to different extents, especially the levels of MMP-9 (P < 0.05). Compared with the BMSCs group, the levels of MMP-2 and MMP-9 in serum as well as activities of MMP-9 and pro-MMP-9 in the brain were decreased significantly in the combined group at 36 and 48 hours after treatment (P < 0.01). The results show that the buyang huanwu decoction can be mutually cooperated with TIMP-1 to inhibit the degradation of extracellular matrix induced by MMP-2 and MMP- 9, repair the damaged blood brain barrier, prevent and cure cerebral edema after ischemia.

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    Changes of cholinergic neurons in the hippocampus of vascular dementia rats after neural stem cell transplantation
    Sun Yu-hua, Geng Li-jiao, Zhao Jing-ya, He Wei-ya, Li Bao-ping
    2015, 19 (50):  8126-8131.  doi: 10.3969/j.issn.2095-4344.2015.50.016
    Abstract ( 272 )   PDF (548KB) ( 459 )   Save

    BACKGROUND: The damage of cholinergic neurons in the frontal cortex and hippocampus in patients with vascular dementia may be the morphological basis of the impairment of cognitive function.
    OBJECTIVE: To investigate the relationship between neural stem cell transplantation and the changes of cholinergic neurons in the hippocampus of rats with vascular dementia.
    METHODS: Ninety Sprague-Dawley rats were randomly divided into three groups, 30 rats in model group, 30 in neural stem cell transplantation group and 30 in sham operated group. Vascular dementia models were established by ligation of the common carotid artery in the model and neural stem cell transplantation groups. The common carotid artery was only separated but not ligated in the sham operated group. After modeling, the neural stem cell transplantation group was injected with 5 μL neural stem cells in the hippocampus CA1 area, while the other two groups were injected the same dose of normal saline. At 3, 7, 14, 30, 60 days after treatment, six rats of each group were sacrificed, respectively. Distributions of BrdU positive cells and ChAT positive cells were detected by S-P immunohistochemical method. The learning and memory abilities of rats were detected by Morris water maze system.
    RESULTS AND CONCLUSION: BrdU positive cells were mainly distributed in the cortex and hippocampus, especially around the blood vessels, and there was the presence of focal aggregation. A small amount of BrdU positive cells were observed in the basal ganglia and thalamus as well as in the ependyma. BrdU positive cells were counted at different time after operation. The number of BrdU positive cells decreased with time, and only a small number of BrdU positive cells were observed at 60 days after transplantation. The number of ChAT positive cells at different time after transplantation was ranked as follows: neural stem cell transplantation group > model group > sham operated group (P < 0.05). Compared with the model group, the time for searching the platform was significantly lower in the neural stem transplantation group and sham operated group, but the number of crossing the platform was significantly higher in the neural stem cell transplantation group and sham operated group (P < 0.05). The results show that neural stem cells could be transplanted into the rats with vascular dementia, and the cells could survive and migrate in the brain of rats and significantly improve the learning and memory ability. This mechanism may be related to the differentiation and growth of cholinergic neurons in the hippocampus. 

     

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    Therapeutic effect of electroacupuncture plus neural stem cell transplantation on the hindlimb function of rats with spinal cord injury 
    Liu Jian-min, Wang Fu-chuan, Zhou Ya-jing, Mu Li, Hou Shao-ke, Hao Li-na, Zhang Zi-tan
    2015, 19 (50):  8132-8138.  doi: 10.3969/j.issn.2095-4344.2015.50.017
    Abstract ( 210 )   PDF (604KB) ( 401 )   Save

    BACKGROUND: Neural stem cell transplantation alone has achieved unsatisfactory outcomes in the repair of damaged spinal cord tissues. To promote the survival, proliferation and neuronal differentiation of transplanted cells in vivo, it is necessary to further improve the micro-environment of spinal cord injury.
    OBJECTIVE: To investigate the effect of neural stem cell transplantation plus electroacupuncture on the hindlimb function and electrophysiological changes of rats with spinal cord injury.
    METHODS: Animal models of spinal cord injury were made in 72 Sprague-Dawley rats and randomized into four groups: control group with injection of culture medium via the tail vein; neural stem cell group with injection of neural stem cell suspension via the tail vein; electroacupuncture group given 1-week electroacupuncture at Du 
    meridian and body points starting from 6 hours after modeling; combined group given injection of neural stem cell suspension via the tail vein+1-week electroacupuncture at Du meridian and body points starting from 6 hours after modeling. Motor functional recovery in rats was assessed by Basso-Beattie-Bresnahan score and inclined plane test before and at 1, 3 days and 1-4 weeks after modeling. At 4 weeks after modeling, hematoxylin-eosin staining was performed for pathological observation; fluorescence microscope was used to observe the survival and distribution of CM-Dil-labeled neural stem cells; horseradish peroxidase tracer was used to observe nerve fiber regeneration; rat neurophysiological recovery was assessed by determining motor evoked potentials and somatosensory evoked potentials.
    RESULTS AND CONCLUSION: At 2-4 weeks after modeling, the hindlimb function was better in the combined group than the neural stem cell group and electroacupuncture group; while it was better in the neural stem cell group and electroacupuncture group than the control group. At 4 weeks after modeling, there were few nerve axon-like structures and small voids in the spinal cord of the neural stem cell group and electroacupuncture group; however, in the combined group, there were more nerve axon-like structures and no void in the spinal cord. At 4 weeks after modeling, the number of nerve fibers positive for CD-Dil and horseradish peroxidase was ranked as follows: combined group > neural stem cell group and electroacupuncture group > control group, and there were significant differences between groups (P < 0.05). The latencies of motor and somatosensory evoked potentials were significantly lower in the combined group than the neural stem cell group and electroaucpuncture group followed by the control group (P < 0.05), whereas the amplitudes of motor and somatosensory evoked potentials were significantly higher in the combined group than the neural stem cell group and electroacupuncture group followed by the control group (P < 0.05). In conclusion, these findings indicate that neural stem cell transplantation combined with electroacupuncture can promote synaptic regeneration and improve the motor and electrophysiological functions of rats.  

     

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    Combination of autologous bone marrow stem cell transplantation and danggui huoxue tang for treatment of diabetic foot
    Ma Hai-tao, Zhou Tao, Qiu Wen-miao, Liu Ping
    2015, 19 (50):  8139-8143.  doi: 10.3969/j.issn.2095-4344.2015.50.018
    Abstract ( 291 )   PDF (502KB) ( 422 )   Save

    BACKGROUND: In recent years, the incidence of diabetic foot is sharply increasing, which tends to cause foot ulcers, amputation and death.
    OBJECTIVE: To investigate the clinical therapeutic effect of autologous bone marrow stem cell transplantation combined with danggui huoxue tang on diabetic foot.
    METHODS: Sixty patients with diabetic foot accompanied by chronic lower limb ischemia were randomly divided into conventional treatment group including 20 cases with 38 affected limbs, and autologous bone marrow stem cell transplantation group (cell transplantation group) including 20 cases with 36 affected limbs, and combined group (autologous bone marrow stem cell transplantation combined with danggui huoxue tang) including 20 cases with 39 affected limbs. After 12 weeks of treatment, indicators such as limb cold-feeling, pain, skin temperature, claudication distance and ankle brachial index were measured, and meanwhile, complications and adverse reactions were observed in the follow-up visit.
    RESULTS AND CONCLUSION: In the conventional treatment group, there was no significant change before and after treatment (P > 0.05). However, these indicators were significantly improved in the other two groups after treatment (P < 0.05) as well as compared with the conventional treatment group (P < 0.05). In addition, there was no adverse reaction in the cell transplantation group. These findings indicate that the combination of autologous bone marrow stem cell transplantation and danggui huoxue tang has effective effects on diabetic foot.  

     

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    Bone marrow mesenchymal stem cell transplantation for liver cirrhosis in rats: variation of liver ultrastructure, stereology parameters and liver function indexes
    Ding Ti-long, Ma Yan-chun, Yu Li, Ma Yong
    2015, 19 (50):  8144-8148.  doi: 10.3969/j.issn.2095-4344.2015.50.019
    Abstract ( 218 )   PDF (580KB) ( 333 )   Save

    BACKGROUND: Mesenchymal stem cell transplantation has a certain controversy in the treatment of liver cirrhosis, and its effects on the receptor liver structure and function need further studies.
    OBJECTIVE: To study the changes in liver ultrastructure, stereology parameters and liver function indexes of rat models with liver cirrhosis treated by bone marrow mesenchymal stem cell transplantation.
    METHODS: Rat models of liver cirrhosis were made using carbon tetrachloride and treated by bone marrow mesenchymal stem cell transplantation. Liver ultrastructure of all the rats were observed by transmission electron microscope, the stereology parameters of the hepatic sinusoid were analyzed by a stereology analysis software, and the serum liver function indexes were detected by a biochemical analyzer.
    RESULTS AND CONCLUSION: (1) The hepatic cells in the rat models exhibited acute hypoxia, lots of mitochondria were destroyed, and obvious karyopycnosis and capillarization of the hepatic sinusoid were found. The liver ultrastructure of rats undergoing cell transplantation was improved remarkably, the hepatic cell nucleus was nearly normal, mitochondrial swelling relieved notably and nuclear pore clogging lessened. (2) The number of hepatic sinusoids in the model group was reduced dramatically, but the total area and mean diameter of the hepatic sinusoid were enlarged significantly as compared with the cell transplantation and normal groups (P < 0.05). (3) Compared with the model group, the albumin level was significantly increased, but the levels of alanine aminotransferase and direct bilirubin were significantly decreased in the cell transplantation (P < 0.05). In addition, 
    there was no significant difference in the level of aspartate aminotransferase between the model and cell transplantation groups (P > 0.05). These findings indicate that bone marrow mesenchymal stem cell transplantation can improve liver function and structure of rats with liver cirrhosis and lessen pathological changes of hepatic sinusoid, so it is an effective treatment for liver cirrhosis. 

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    Heat shock improves Sca-1+ cell transplantation for treatment of myocardial infarction in mice
    Wang Li-hui, Shen Ya-hui, Guo Yan-qing, Zang Bin-bin, Li Wei
    2015, 19 (50):  8149-8154.  doi: 10.3969/j.issn.2095-4344.2015.50.020
    Abstract ( 246 )   PDF (522KB) ( 449 )   Save

    BACKGROUND: Recent studies have found that stem cells can directly differentiate into mature myocardial cells or promote their regeneration, providing a new therapeutic strategy for the treatment of myocardial infarction. However, the low cell transplantation rate reduces the myocardial differentiation ability and myocardial repair.
    OBJECTIVE: To study the role of heat shock treatment in Sca-1+ cell transplantation for treatment of myocardial infarction in mice.
    METHODS: Sca-1+ cells were isolated from the bone marrow of mice using magnetic bead sorting method, and were subjected to heat shock treatment. Animal models of myocardial infarction were made in mice, and then randomized into two groups: heat shock group and non-heat shock group, which were given 1 mL heat shock-treated Sca-1+ cells and 1 mL non-heat shock-treated Sca-1+ cells via the tail vein, respectively. After 
    transplantation, cell survival, heart function, myocardial cell apoptosis and myocardial fibrosis were detected. Meanwhile, the expressions of heat shock factor (HSP), HSP70 and miR-34a in the left ventricle were measured.
    RESULTS AND CONCLUSION: (1) The expression of sry gene in the heat shock group was significantly higher than that in the non-heat shock group. (2) The left ventricular ejection fraction and fractional shortening in the heat shock group were significantly higher than those in the non-heat shock group. The left ventricular end-diastolic diameter and systolic diameter in the heat shock group were significantly lower than those in the non-heat shock group. (3) The cardiac fibrosis and myocardial cell apoptosis in the heat shock group were significantly lower than those in the non-heat shock group. (4) The HSF and HSP70 expression in the left ventricle was significantly higher in the heat shock group than the non-heat shock group, and the miR-34a expression in the left ventricle was significantly lower in the heat shock group than the non-heat shock group. These findings indicate that heat shock-treated Sca-1+ cell transplantation can reduce myocardial apoptosis and infarct size, and improve heart function of mice with myocardial infarction. 

     

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    Derivation of mesenchymal stem cell lines from menstrual blood
    Tan Ji-chun, Li Ya-xuan, Wang Qiu-shi, Li Xiao-ni
    2015, 19 (50):  8155-8160.  doi: 10.3969/j.issn.2095-4344.2015.50.021
    Abstract ( 471 )   PDF (730KB) ( 820 )   Save

    BACKGROUND: Menstrual blood-derived mesenchymal stem cells are proved to be a new type of adult stem cells. To date, little is reported on the methods of establishing this kind of cell lines systematically.
    OBJECTIVE: To isolate, culture, identify and finally establish menstrual blood-derived mesenchymal stem cell lines.
    METHODS: Menstrual blood-derived mesenchymal stem cells were isolated from the menstrual blood of healthy female donors and cultured accordingly using density gradient centrifugation method. Cell morphology and proliferation characteristics were observed, and proliferative ability was detected by cell counting kit-8 assay. Cells were collected periodically for karyotype analysis. Expressions of cell surface antigens such as CD34, CD38, CD44, CD45, CD73, CD90, CD105 and Oct-4 were analyzed using flow cytometry. Cell pluripotency were identified by inducing adipogenesis and osteogenesis in vitro.
    RESULTS AND CONCLUSION: We successfully established four menstrual blood-derived mesenchymal stem cell lines from the menstrual blood of six healthy volunteers. The cultured cell lines had a typical spindle shape and kept normal 46, XX karyotype. The cells grew rapidly and entered into the logarithmic growth phase at 48 hours after passaging. The results of flow cytometry showed that menstrual blood-derived mesenchymal stem cells were positive for CD44, CD73, CD90, CD105 and Oct-4, while negative for CD38, CD34 and CD45. Menstrual blood-derived mesenchymal stem cells were positive for oil red O staining at 22 days after adipogenic 
    induction and also positive for alizarin red staining at 2 weeks after osteogenetic induction. In conclusion, our study suggests that menstrual blood-derived mesenchymal stem cells with stable karyotype and pluripotency can proliferate quickly, and we can provide a new resource for stem cell therapy and regenerative medicine through establishing the menstrual blood-derived mesenchymal stem cell lines.  

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    Spinal cord-derived neural stem cells cultured by serum-free suspension method: separation, cultivation and identification
    Li Jun, Xu Da-bo, Fu Qiang, Liu Yan-bin
    2015, 19 (50):  8161-8166.  doi: 10.3969/j.issn.2095-4344.2015.50.022
    Abstract ( 267 )   PDF (557KB) ( 437 )   Save

    BACKGROUND: Until now, there is yet no complete recovery from spinal cord injury in terms of structure and functional recoveries. Neurotrophic factors have limited effects on nerve regeneration. Currently, stem cell transplantation may be an effective way to repair spinal cord injury.
    OBJECTIVE: To separate, cultivate and purify mouse spinal cord-derived neural stem cells using serum-free suspension method followed by morphological observation, immunofluorescence technology and multi-lineage differentiation experiments.
    METHODS: By using the suspension culture method, mouse spinal cord-derived neural stem cells at embryonic day 13.5 were cultured and purified. Cell morphology changes were observed under inverted microscope. Cell proliferation ability was detected using cell counting kit-8. Nestin and Sox2 expression was detected by immunofluorescence technology. Multilineage differentiation of spinal cord-derived neural stem cells at passage 4 was detected by natural differentiation method in order to prove the differentiation ability.
    RESULTS AND CONCLUSION: Serum-free medium suspension culture method was successfully applied to separate spinal cord-derived neural stem cells. Cultured cells had good proliferative ability and highly expressed Nestin and Sox2 that was in accordance with the results of DAPI nucleus staining, suggesting the high purity of cells. After induction, the cells could express both Tuj1 and GFAP, indicating the cells had good differentiation 
    potential. This experiment has successfully established the isolation, culture, identification system of spinal cord-derived neural stem cells, providing experimental basis for subsequent studies of neural stem cells. 

     

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    Differentiation of neural stem cells induced by glial cell line-derived neurotrophic factor
    He Jia, Yan Bo
    2015, 19 (50):  8167-8171.  doi: 10.3969/j.issn.2095-4344.2015.50.023
    Abstract ( 270 )   PDF (481KB) ( 550 )   Save

    BACKGROUND: The use of neural stem cells provides a new approach for nervous system functional reconstruction and nerve regeneration. How to solve the induced differentiation of neural stem cells is still a research hotspot.
    OBJECTIVE: To investigate the differentiation of rat neural stem cells into neurons and dopaminergic neurons under induction of glial cell-line derived neurotrophic factor gene.
    METHODS: PcDNA3-GDNF-GFP plasmids were constructed and transferred via lipidosome into rat embryonic neural stem cells. Differentiation of neural stem cells after transfection was identified under a fluorescence microscope. Immunofluorescent staining was used to detect expression of β-tubulin III and tyrosine hydroxylase.
    RESULTS AND CONCLUSION: At 3 days after transfection, green fluorescence was observed in the transfected cells that were globular. At 7 days after transfection, the proportion of neural stem cells differentiating into neurons and dopaminergic neurons were significantly increased. These results indicate that the glial cell-line derived neurotrophic factor gene can induce the differentiation of neural stem cells into neurons and dopaminergic neurons. 
     

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    Neuronal differentiation of cell subsets with stem cell characteristics in adult rat meningeal tissues
    Liu De, Li Xiang-ming, Gu Xi-juan
    2015, 19 (50):  8172-8176.  doi: 10.3969/j.issn.2095-4344.2015.50.024
    Abstract ( 265 )   PDF (1KB) ( 303 )   Save

    BACKGROUND: Neural stem cells have the potential to differentiate into neurons and glial cells to replace the injured brain cells, so as to achieve the purpose of repairing nerve injury.
    OBJECTIVE: To observe the neuronal differentiation ability of cell subsets with stem cell characteristics in the adult rat meningeal tissues.
    METHODS: Under anesthesia, the meningeal tissues were obtained from adult Sprague-Dawley rats to make cell suspension followed by inoculation and subculture. Then, the Nestin immunofluorescence staining was performed. The third generation cells were cultured in vitro with complete culture medium containing trichostatin A. After 7 days of induction, western blot assay was used to detect the expression of NF-200 and BM88 proteins in neural cells.
    RESULTS AND CONCLUSION: At 24 hours of culture, some spherical cells were suspended and some cells adherent. In addition, some spherical cells scattered gradually formed the clone spheres, and the growth rate decreased with the increasing volume. The positive expression of Nestin was detected by immunocytochemistry staining, and the cell nucleus was stained blue by Hoechst staining. BM88 and NF-200 proteins were all expressed at 7 days of neural induction. These findings indicate that the cell subsets with stem cell characteristics in the adult rat meningeal tissues can differentiate into neurons after in vitro induction. 
     

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    Effects of soft substrates on the chondrogenic differentiation of human synovial-derived mesenchymal stem cells
    Cui Shuang-shuang, Yu Zhao-zhen, Yu Shun-lu, Zhao Wen-jun, Zhao Li-kun, Xing Guo-sheng, Duan Xiao-yuan
    2015, 19 (50):  8177-8183.  doi: 10.3969/j.issn.2095-4344.2015.50.025
    Abstract ( 290 )   PDF (637KB) ( 454 )   Save

    BACKGROUND: Our previous studies have shown that a soft substrate has a significant effect on morphology and cytoskeleton of rat bone marrow mesenchymal stem cell.
    OBJECTIVE: To explore the effect of polyacrylamide gels as soft substrates with different elastic moduli on the chondrogenic differentiation of human synovial-derived mesenchymal stem cells.
    METHODS: The synovium was harvested from patients with osteoarthritis under sterile conditions, and primary human synovial-derived mesenchymal stem cells were separated using limiting dilution assay. The flow cytometry and multi-directional differentiation experiments were used to identify the cell surface markers and function of the human synovial-derived mesenchymal stem cells, respectively. The polyacrylamide gels with the elastic modulus of 0.4, 6, 30 kPa, which were made using various amounts of acrylamide and bis-acrylamide, were used to culture human synovial-derived mesenchymal stem cells under induction with transforming growth 
    factor-β1 for 7 and 14 days. RT-PCR was used to test the expression of chondrogenic genes, type II collagen gene and cartilage acidic protein 1. The 6-well cell culture plates served as controls.
    RESULTS AND CONCLUSION: The human synovial-derived mesenchymal stem cells showed different cell morphology in the different elastic modulus of polyacrylamide gels. The expression of type II collagen gene and cartilage acidic protein 1 were affected by the different elastic modulus of polyacrylamide gels and culture time, and there was an interaction between these two factors. At 7 days of induction, the expression of cartilage acidic protein 1 gene on 6 kPa polyacrylamide gels was the highest (F=44.350, P=0.000); meanwhile, the expression of type II collagen gene on 0.4 kPa polyacrylamide gels was the highest (F=6.384, P=0.005). These findings indicate that polyacrylamide gels with lower elastic modulus are superior to routine culture plates to promote the chondrogenic differentiation of human synovial-derived mesenchymal stem cells. 

     

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    Analysis and identification of a novel CD36 allele, 1142 T>G
    Lin Feng-qiu, Li Xiao-feng, Shao Chao-peng, Li Jian-ping
    2015, 19 (50):  8184-8189.  doi: 10.3969/j.issn.2095-4344.2015.50.026
    Abstract ( 280 )   PDF (504KB) ( 468 )   Save

    BACKGROUND: As a main antigen of platelet, CD36 antigen is also known as platelet glycoprotein IV (GPIV). The mutation of CD36 gene may result in deficiency of the antigen.
    OBJECTIVE: To identify a novel CD36 allele.
    METHODS: DNA was isolated from peripheral blood sample, and 12 coding regions of CD36 gene were amplified by PCR. Sequencing-based typing was used to analyze the sequence of the target regions. The derived sequences were aligned with the standard sequence of NG_008192 in GenBank to identify the novel allele.
    RESULTS AND CONCLUSION: 1142 T>G mutation was detected in exon 12 of CD36 gene of the proband, and the other regions were consistent with the standard sequence. No data or report about 1142 T>G was found in GenBank or National Center for Biotechnology Information (NCBI), and thus it was reported to GenBank and received by number KM275213. 1142 T>G results in amino acid 381 Leu>Ser of the CD36 protein. There is a big
    difference in hydrophilia and polarity of the two amino acids. Also the 381 amino acid locates in highly conserved region. Thus it is speculated that 1142 T>G may reduce or vanish the activity of the protein.  

     

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    Effect of human placenta-derived mesenchymal stem cells on cytotrophoblast apoptosis in gestational hypertension: study protocol for a randomized controlled trial
    Zhang Li, Li Zhi, Wang Jing, Liu Wei
    2015, 19 (50):  8190-8194.  doi: 10.3969/j.issn.2095-4344.2015.50.027
    Abstract ( 257 )   PDF (463KB) ( 462 )   Save

    BACKGROUND: Excessive apoptosis and decreased infiltration of cytotrophoblasts are essential causes for hypertension in pregnancy. Human placenta-derived mesenchymal stem cells contribute to damage repair, which has been shown in many studies, and moreover, human placenta-derived mesenchymal stem cells have a certain endocrine function and can act on the other tissues in an autocrine or paracrine manner. Therefore, we attempt to explore whether the human placenta-derived mesenchymal stem cell can repair damaged cytotrophoblasts, and then to gestational hypertension.
     
    METHODS/DESIGN: This is a randomized controlled cytological experiment. Human placenta-derived mesenchymal stem cell culture medium is collected and filtrated as human placenta-derived mesenchymal stem cell conditioned medium. Human cytotrophoblasts, JEG-3 cells, are cultured and randomized into three groups:15% normal pregnant serum is added in control group; 15% serum from severe pre-eclampsia patients is added in gestational hypertension model group; and in condition medium group, 15% serum from severe pre-eclampsia patients is added for 24 hours of culture, and then human placenta-derived mesenchymal stem cell conditioned medium containing 15% serum from severe pre-eclampsia patients is used instead. Flow cytometry is used to detect cell apoptosis rate.
    DISCUSSION: This study will help to find the feasibility of cell transplantation for gestational hypertension by exploring the effect of human placenta-derived mesenchymal stem cells on cytotrophoblasts function in gestational hypertension.
    ETHICAL APPROVAL: The protocol is approved by the Ethics Committee of Shenyang Central Hospital of Shenyang Medical University. Informed consent is obtained from normal and pre-eclampsia women in pregnancy. 
     

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    Mesenchymal stem cells promote articular cartilage repair and regeneration
    Zhu Yu-qi, Wang Jin-rong, Wang Zhi-yao
    2015, 19 (50):  8195-8200.  doi: 10.3969/j.issn.2095-4344.2015.50.028
    Abstract ( 338 )   PDF (570KB) ( 549 )   Save

    BACKGROUND: After articular cartilage injury, the injured cartilage almost has no self-healing ability. Articular cartilage injury repair has been always a difficulty in clinical work.
    OBJECTIVE: To explore the types and biological characteristics of stem cells for articular cartilage repair and to ensure the role and relative merits of stem cell transplantation in articular cartilage repair.
    METHODS: PubMed and CNKI were retrieved by the first author for relevant articles published from 1998 to 2015 using the keywords of “articular cartilage injury, mesenchymal stem cells, regeneration” in English and Chinese, respectively. Finally, 47 articles were included in result analysis.
    RESULTS AND CONCLUSION: Stem cell therapy is the most effective method for repair of articular cartilage injury. Mesenchymal stem cells from bone marrow, adipose and umbilical cord have strong chondrogenic and cloning capacities. Bone marrow mesenchymal stem cells have a stronger differentiation potential, and can be used for repair of cartilage injury. Umbilical cord-derived mesenchymal stem cells have a low tumorigenicity. Adipose-derived stem cells can proliferate and grow faster. Stem cells combined with natural carrier materials, such as collagen, gelatin, fibrin and alginate, can promote cell adhesion, differentiation and proliferation, in order to build an effective tissue engineered cartilage for repair of articular cartilage defects. 

     

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