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18 October 2017, Volume 21 Issue 29 Previous Issue    Next Issue

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LncRNA-Braveheart promotes the differentiation of bone marrow mesenchymal stem cells in vitro into cardiomyocyte-like cells Hou Jing-ying, Long Hui-bao, Zhou Chang-qing, Wu Hao, Guo Tian-zhu, Zhong Ting-ting, Wu Quan-hua, Wang Lei, Zheng Shao-xin, Wang Tong 2017, 21 (29):  4593-4599.  doi: 10.3969/j.issn.2095-4344.2017.29.001 Abstract ( 370 )   PDF (1436KB) ( 729 )   Save BACKGROUND: Our previous work demonstrated that bone marrow mesenchymal stem cells (BMSCs) transplantation could improve cardiac function in rats with myocardial infarction. However, the overall efficacy was unsatisfactory, and there was a low efficiency of BMSCs differentiating into cardiomyocytes in the local infarct myocardium. OBJECTIVE: To transfect long non-coding RNA-Braveheart (lncRNA-Bvht) into BMSCs in order to observe whether it could promote cardiomyocyte differentiation of BMSCs in vitro. METHODS: pLVX-IRES-ZsGreen1-lncRNA-Bvht vector was constructed and applied to transfect lncRNA-Bvht into bMSCs, and then, the transfection efficiency was detected. BMSCs were obtained from C57BL/6 mice and cultured in vitro. Passage 3 cells were divided into three groups: BMSCs group, null vector group and lncRNA-Bvht group. All cells in the three groups were cultured in the normal condition for 48 hours and cardiomyocytes differentiation was induced by 5-azacytidine for another 24 hours followed by 2-week culture under normal conditions. Cardiomyocyte differentiation of BMSCs was observed under fluorescence microscopy and expression of cardiac specific cell markers including troponin T and myosin were examined using immunofluorescent staining, western blot assay, and qRT-PCR. RESULTS AND CONCLUSION: Cell morphological changes could be observed in all the groups 2 weeks after the induction. Interconnected cells arranged consistently in all the three groups. Immunofluorescent staining results showed that the expression of troponin T and myosin was notably positive, and the proportion of troponin T positive cells was significantly increased. qRT-PCR and western blot assay results indicated that there were significantly increased levels of troponin T and myosin in the lncRNA-Bvht group as compared with the BMSCs and null vector groups (P < 0.01), suggesting that lncRNA-Bvht could efficiently promote cardiomyocyte differentiation of BMSCs in vitro.  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 Figures and Tables | References | Related Articles | Metrics

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Effects of parathyroid hormone on calcium ions in rat bone marrow mesenchymal stem cells Chen Yu-shu, Zhang Yan-hong, Zhang Shu-jiang, Bai Bo, Chen Yi 2017, 21 (29):  4600-4604.  doi: 10.3969/j.issn.2095-4344.2017.29.002 Abstract ( 299 )   PDF (1284KB) ( 190 )   Save BACKGROUND: Parathyroid hormone can activate a series of physiological and biochemical reactions through specific receptors on the surface of target organs, to adjust the dynamic calcium balance. OBJECTIVE: To explore the mechanisms whereby parathyroid hormone maintains in vitro proliferation of bone marrow mesenchymal stem cells (BMSCs), by observing the effects of parathyroid hormone on calcium ions in BMSCs. METHODS: BMSCs at passage 4 were randomly divided into two groups. Experimental group was treated with parathyroid hormone, and control group was treated with routine culture medium. During 10 days of culture, MTT assay was used to detect BMSCs proliferation. At 7 and 14 days after treatment, laser scanning confocal microscopy was used to detect fluorescence intensity of calcium ions in BMSCs in the two groups. RESULTS AND CONCLUSION: (1) At 1 and 2 days after culture, the cell viability in the two groups was sluggish. At 3 days after culture, the cells turned into the rapid growth. At 7 and 8 days after culture, the cell growth turned into the plateau. At 10 days after culture, the cell proliferation ability had a weakening trend, which was lower in the control group than in the experimental group (P < 0.05). (2) At 7 days after culture, the fluorescence intensity of intracellular calcium ions was strong. As the induction time extended, the fluorescence intensity of intracellular calcium ions was declined at 14 days after-culture. At 7 and 14 days after culture, the fluorescence intensity of intracellular calcium ions in the experimental group was higher than that in the control group (P < 0.001). There was no difference in the fluorescence intensity of intracellular calcium ions between experimental group at 14 days and control group at 7 days. To conclude, parathyroid hormone could increase the fluorescence intensity of calcium ions in BMSCs, and function to maintain the BMSCs proliferation.   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 Figures and Tables | References | Related Articles | Metrics

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Tetracycline hydrochloride induces the osteogenic differentiation of rat bone marrow mesenchymal stem cells Zhang Jue, Xue Si-liang, Luo Yuan, Zhi Wei 2017, 21 (29):  4605-4610.  doi: 10.3969/j.issn.2095-4344.2017.29.003 Abstract ( 303 )   PDF (4307KB) ( 548 )   Save BACKGROUND: Tetracycline hydrochloride not only promotes the proliferation of bone marrow mesenchymal stem cells (BMSCs), but also has a strong bone activity. Studies on the osteogenic differentiation of BMSCs induced by tetracycline hydrochloride will provide a new cell source for bone tissue engineering. OBJECTIVE: To observe the induction and differentiation activity of BMSCs into osteoblasts cultured by tetracycline hydrochloride in vitro. METHODS: BMSCs were separated and expanded by whole bone marrow adherent method and identified by cell morphology and BMSCs cell markers CD34, CD45, CD90, CD29. Passage 3 BMSCs were collected and cultured with tetracycline hydrochloride (experimental group) or cultured in L-DMEM containing 10% fetal bovine serum (control group). Osteogenic induction and differentiation activity of BMSCs cultured by tetracycline hydrochloride were detected by alkaline phosphatase activity detection, collagen I and osteocalcin immunohistochemistry staining, alizarin red staining of mineralized calcium nodules, and detection of collagen I and osteocalcin gene expression by RT-PCR. RESULTS AND CONCLUSION: Tetracycline hydrochloride continuously promoted alkaline phosphatase activity in the BMSCs, significantly different from that in the control group. After 21 days of culture, positive expression of collagen I and osteocalcin proteins, red calcium nodules shown by alizarin red staining, and mRNA expression of collagen I and osteocalcin were observed in the experimental group, while the negative expression was found in the control group. In conclusion, tetracycline hydrochloride has a significant role to promote the osteogenic differentiation of BMSCs. 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 Figures and Tables | References | Related Articles | Metrics

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Bone marrow mesenchymal stem cell transplantation for skin wound healing Zhang Bei-ying, Luo Dong-zhang, Mai Hai-tao, Chen Xiang-lang, Guo Yan, Wang Bing-yun, Chen Sheng-feng, Chen Zhi-sheng 2017, 21 (29):  4611-4616.  doi: 10.3969/j.issn.2095-4344.2017.29.004 Abstract ( 379 )   PDF (4267KB) ( 225 )   Save BACKGROUND: At present, most of the experiments on the treatment of skin wound by mesenchymal stem cells have been performed in rats, mice and rabbits, while the research on skin wound treatment by canine bone marrow mesenchymal stem cells is less reported. OBJECTIVE: To observe the effect of canine bone marrow mesenchymal stem cells on skin wound healing. METHODS: A 3 cm×3 cm wound was made on the both sides of the scapula and buttocks of the dog, with the right side as experimental group and the left side as control group. After the wound was made, allogeneic canine bone marrow mesenchymal stem cells suspension was injected subcutaneously around the wound in the experimental group on the 1st and 3rd days. The control group was injected subcutaneously around the wound with mesenchymal stem cell culture medium on the 1st and 3rd days after the wound was made. Wound healing was observed dynamically in both groups. RESULTS AND CONCLUSION: In the 1st week, there were pale yellow inflammatory substances in the wound of two groups indicating obvious inflammations. Compared with the control group, the inflammatory substances were fewer and the growth rate of the granulation tissue was faster in the experimental group. From the 2nd week until the wound healing, epithelialization on the wound became obvious following the formation of the granulation tissue, which was mainly displayed by the formation of fresh epithelial tissues from the surrounding to the wound center. The epithelialization time of the experimental group was earlier than that of the control group, and the wound area of the experimental group was smaller than that of the control group. In the 3rd week, the wound in the experimental group healed completely, and became smoother than that in the control group. The wound area of the experimental group was slightly smaller than that of the control group on the 8th and 12th days after cell transplantation, and the healing speed of the experimental group was slightly faster than that of the control group, but there was no significant difference between the two groups. Our findings indicate that the transplantation of canine bone marrow mesenchymal stem cells has the possibility or trend to promote skin wound healing. 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 Figures and Tables | References | Related Articles | Metrics

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Tolerance of rat bone marrow mesenchymal stem cells overexpressing human heme oxygenase 1 to ischemia/hypoxia injury Deng Ning-bo, Han Teng-long, Zeng Yuan-qing, Jiang Zhi-xin 2017, 21 (29):  4617-4622.  doi: 10.3969/j.issn.2095-4344.2017.29.005 Abstract ( 371 )   PDF (4323KB) ( 176 )   Save BACKGROUND: Under ischemia/hypoxia microenvironment, very low survival rate of transplanted bone marrow mesenchymal stem cells (BMSCs) in the host limits its efficacy in the treatment of acute myocardial infarction. OBJECTIVE: To explore the tolerance of human heme oxygenase 1 (hHO-1) gene modified rat BMSCs to ischemia/hypoxia injury.   METHODS: The rat BMSCs were transfected with hHO-1 recombinant adenovirus. Western blot assay was used to determinate the optimal time of hHO-1 protein expression. hHO-1 modified rat BMSCs were cultured in hypoxia and serum-free conditions that simulated ischemia/hypoxia microenvironment in vivo. Cell counting kit-8 and trypan blue staining were performed to detect the survival rate of BMSCs at 12, 24, 48, 72 hours after culture under the ischemia/hypoxia microenvironment. Flow cytometry was used to detect apoptosis in BMSCs at 24 hours after culture under the ischemia/hypoxia microenvironment.  RESULTS AND CONCLUSION: The expression of hHO-1 protein was highest at 4 days after transfection. Under the ischemia/hypoxia microenvironment for 12, 24, 48, 72 hours, the survival rates of transfected BMSCs were significantly higher as compared with the untransfected cells (P < 0.05), shown by the cell counting kit-8 and trypan blue staining. In addition, the results from flow cytometry showed that there was a higher survival rate of transfected BMSCs than untransfected cells at 24 hours of culture under the ischemia/hypoxia microenvironment (P < 0.05). To conclude, hHO-1 modified rat BMSCs have stronger tolerance to the ischemia/hypoxia microenvironment. 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 Figures and Tables | References | Related Articles | Metrics

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Culture and identification of rhesus monkey bone marrow mesenchymal stem cells Dong Jing-jing, Hu Zhu-lin, Li Yan, Sun Xiao-mei 2017, 21 (29):  4623-4628.  doi: 10.3969/j.issn.2095-4344.2017.29.006 Abstract ( 344 )   PDF (1473KB) ( 212 )   Save BACKGROUND: Studies on rhesus monkey bone marrow mesenchymal stem cells (BMSCs) are limited, and the differentiation potential of juvenile rhesus monkey BMSCs remains unknown. OBJECTIVE: To investigate the biological characteristics of rhesus monkey BMSCs isolated and cultured in vitro,  and to identify the growth curve and phenotype of BMSCs. METHODS: BMSCs from juvenile rhesus monkeys were isolated and cultured by density gradient centrifugation and the whole bone marrow adherence methods. BMSCs at passages 2 and 7 were taken to draw cell growth curves by MTS method. Flow cytometry was used to detect apoptosis of passage 3 BMSCs and to detect expression of cell surface markers, CD29, CD34, CD45, CD90, CD147, in passage 4 BMSCs. Osteogenic and adipogenic abilities of passage 3 BMSCs were also detected. RESULTS AND CONCLUSION: The BMSCs at passages 2 and 7 were incubated at first 2 days, logarithmically grew at 3-7 days, and then proliferated slowly at 8 days. The BMSCs at passage 2 showed stronger proliferation than those at passage 7. At passage 3, BMSCs were negative for CD34 and CD45, but positive for CD90, CD29 and CD147. Results from the flow cytometry showed that there were 73.83% living cells, 21.76% dead cells, and 4.27% apoptotic cells. BMSCs could differentiate into osteoblasts and adipocytes under osteogenic and adipogenic induction, respectively. To conclude, BMSCs from the rhesus monkey have strong proliferation and multilineage differentiation abilities in vitro.  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 Figures and Tables | References | Related Articles | Metrics

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5-Azacytidine combined with angiotensin II induces differentiation of  adipose-derived mesenchymal stem cells into cardiomyocytes Wang Jing, Han Jiang-hong, Li Qiong 2017, 21 (29):  4629-4634.  doi: 10.3969/j.issn.2095-4344.2017.29.007 Abstract ( 433 )   PDF (1136KB) ( 173 )   Save BACKGROUND: Taking effective measures for myocardial necrosis develops a trend in the clinical treatment of myocardial infarction. OBJECTIVE: To investigate the mechanism of 5-azacytidine combined with angiotensin II in the differentiation of mouse adipose-derived mesenchymal stem cells into cardiomyocytes. METHODS: Twenty ICR mice aged 4 weeks old were used to isolate adipose-derived mesenchymal stem cells using type I collagenase digestion method. Cell morphology was observed, cell proliferation was detected using MTT, and immunofluorescence technique was used to detect the expression of CD90, CD105, CD73 and CD45. Passage 4 adipose-derived mesenchymal stem cells were cultured for 24 hours, then induced by 15 μmol/L 5-azacytidine or  15 μmol/L 5-azacytidine plus 0.1 μmol/L angiotensin II for another 24 hours, and finally cultured in a complete medium. The expression of cTnT protein in myocardial cells was detected by western blot assay. RESULTS AND CONCLUSION: Primary adipose-derived mesenchymal stem cells with small nuclei were spindle-shaped or starlike. After passage, the cells were increased in size, with uniform shape and regular arrangement. Passage 3 mouse adipose-derived mesenchymal stem cells showed an S-shaped growth curve: days 1-3 was incubation period, the cells showed logarithmic growth at day 4, the cell growth reached the peak at days 5-7, and then entered stationary phase. The passage 4 cells were positive for CD90, CD105 and CD73, but negative for CD45. The expression of cTnT was significantly higher after cell induction with 5-azacytidine combined with angiotensin II than 5-azacytidine alone. In conclusion, the combined use of 5-azacytidine and angiotensin II is beneficial for the cardiomyocyte-like differentiation of adipose-derived 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 Figures and Tables | References | Related Articles | Metrics

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Effects of epidermal growth factors on the proliferation and migration of rat bone marrow mesenchymal stem cells Xia Rong-mu, Jiang Qi-chang, Yang Long, Xie Tian-hong, Lu Hong-ling, Jiang Zheng-ju 2017, 21 (29):  4635-4641.  doi: 10.3969/j.issn.2095-4344.2017.29.008 Abstract ( 371 )   PDF (5001KB) ( 238 )   Save BACKGROUND: Epidermal growth factor is an auxiliary growth factor, but its effect on the growth of bone marrow mesenchymal stem cells (BMSCs) is uncertain. OBJECTIVE: To establish a mature method for isolation, extraction and identification of rat BMSCs, to investigate the effects of epidermal growth factor (EGF) on the proliferation and migration ability of BMSCs and to explore its potential mechanisms at the same time. METHODS: Rat BMSCs were isolated and purified using the improved whole bone marrow adherence method. After the cells were subcultured to the third generation, we detected the expression of cells surface antigens CD29, CD45 and CD90 by flow cytometry. BMSCs were further identified by osteogenic and adipogenic differentiation. Meanwhile, the effect of EGF on the proliferation of passage 3 BMSCs was measured by cell counting kit-8 and clonogenic assay. And the migration of P3 cells was verified by Transwell chamber. In addition, we detected the expression of proteins related to PI3K/Akt and nuclear factor-κB signaling pathways by western blot assay. RESULTS AND CONCLUSION: The primary BMSCs were polygonal and spindle-shaped, and then gradually appeared to be spindle-shaped. The results of flow cytometry demonstrated that the passage 3 cells were positive for CD29 and CD90, but negative for CD45. Furthermore, we successfully induced the osteogenic and adipogenic differentiation of BMSCs in vitro. Additionally, our data demonstrated that EGF promoted the proliferation and migration of passage 3 BMSCs. The relative expression levels of p-Akt and Bcl-2 of PI3K/Akt signaling pathway was up-regulated and the expression of Bax was down-regulated. At the same time, the relative expression level of phosphorylated p65 of nuclear factor-κB signaling pathway was up-regulated and the expression of phosphorylated inhibitor κB was down-regulated. Moreover, the downstream protein of matrix metalloproteinase-9 was up-regulated. Those proteins were related to the migaration of BMSCs. In summary, our results suggest that EGF could promote the proliferation and migration of BMSCs. 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 Figures and Tables | References | Related Articles | Metrics

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Transfection efficiency of three different lentiviruses with green fluorescent protein gene to transfect rabbit bone marrow mesenchymal stem cells Xiao Yu, Li Yan-lin, Wang Kun, Li Long-teng, Yan Xiang-jia, Meng Xu-han 2017, 21 (29):  4642-4647.  doi: 10.3969/j.issn.2095-4344.2017.29.009 Abstract ( 394 )   PDF (5113KB) ( 361 )   Save BACKGROUND: We explore the transfection effects of different lentiviruses on bone marrow mesenchymal stem cells (BMSCs) from the same source in order to screen out appropriate cell transfection tools. OBJECTIVE: To explore the difference in the transfection efficiency of three different lentiviruses carrying green fluorescent protein (GFP) to transfect the rabbit BMSCs. METHODS: Bone marrow samples were extracted from the ilium of 2-week-old Japan white rabbits, and then the BMSCs were separated, purified and extended using density gradient centrifugation. Cell surface markers were identified using flow cytometry. pTomo-GFP lentivirus, Ad-GFP lentivirus and lenti-GFP lentivirus were used to transfect the BMSCs, and the transfection efficiency was tested using flow cytometry. The cell viability was detected using cell counting kit-8 before and after transfection. RESULTS AND CONCLUSION: As the positive rates of CD44, CD34 and CD45 were 75.64%, 6.14% and 9.48%, respectively, the cultured cells were identified as BMSCs by the flow cytometry. The expression levels of GFP after transfection by pTomo-GFP, Ad-GFP and lenti-GFP were 2.64%, 16.72% and 45.24%, respectively. The cell growth curve showed that pTomo-GFP lentivirus, Ad-GFP and lenti-GFP lentivirus showed certain effects on cell growth. pTomo-GFP and Ad-GFP had an effect on cell growth at incubation period and logarithmic growth phase at 1-8 days after transfection, which may cause some interference to the viability of early cells. lenti-GFP had little effect on cell growth in the short term, but there was a change in cell growth at day 12 after lenti-GFP transfection, and a long-term effect of lenti-GFP on cell viability was considered. Therefore, lenti-GFP lentivirus could highly transfect the BMSCs and could not impact the normal growth of BMSCs, indicating that lenti-GFP lentivirus is suitable for BMSCs tracing and gene transfection. 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 Figures and Tables | References | Related Articles | Metrics

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Biological characteristics of side population cells in multiple myeloma U266 cell line in relation to drug resistance Bao Qiu-ping, Li Hui-min, Li Xiao-jin, Yan Yan-hong 2017, 21 (29):  4648-4653.  doi: 10.3969/j.issn.2095-4344.2017.29.010 Abstract ( 300 )   PDF (4700KB) ( 277 )   Save BACKGROUND: Studies have shown that tumor stem cells are the basis and the main cause of tumor recurrence and chemotherapy failure. Therefore, the research on the drug resistance of tumor stem cells has become a hotspot in the field of stem cell research. OBJECTIVE: To analyze the biological characteristics of side population (SP) cells in the U266 cell lines in relation to drug resistance. METHODS: Flow cytometry was used to monitor the percentage of SP cells in the U266 cell lines. Fluorescence-activated cell sorter (FACS) analysis was used to isolate SP and non-SP (NSP) cells from the U266 cell lines. Further analyses of the cell cycle, multidrug resistant protein, methyl cellulose cloning assay of SP cells and NSP cells were performed. Real-time quantitative PCR was used to determine the expression of ABCG2 and MDR1 genes. RESULTS AND CONCLUSION: There were (2.46±0.35)% SP cells in U266 cells, which were most in the G0 phase. The ratio of G0/G1 in SP cells was (81.50±5.42)%, which was significantly higher than that in NSP cells [(39.85±3.21)%; P < 0.05]. The positive expression rates of P-gp and ABCG2 in SP cells were signficantly higher than those in NSP cells (P < 0.05). The cloning efficiency of SP cells was significantly higher than that of NSP cells (P < 0.05). The mRNA expression of ABCG2 and MDR1 was also significantly higher in SP cells than in NSP cells (P < 0.05). To conclude, a small subpopulation of the isolates of U266 cell lines belong to tumor stem cell-like SP cell subset, most of which are at the G0 phase. ABCG2 and MDR1 genes are highly expressed in SP cells, which particularly plays a important role in the multidrug resistance of multiple myeloma stem cell lines. 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 Figures and Tables | References | Related Articles | Metrics

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A comparative study on two culture methods for adipose-derived stem cells from mice with osteoporosis Huang Cheng-long, Li Qing, Wang Lei, Huang Kui, Luo Dao-wen, Xiao Jin-gang 2017, 21 (29):  4654-4659.  doi: 10.3969/j.issn.2095-4344.2017.29.011 Abstract ( 308 )   PDF (1774KB) ( 236 )   Save BACKGROUND: To obtain enough adipose-derived stem cells (ADSCs) is the premise of repairing osteoporotic bone defects by autograft transplantation; therefore, how to efficiently, simply and economically harvest primary autologous ADSCs is the key to the treatment of osteoporosis. OBJECTIVE: To isolate and culture ADSCs from mice with osteoporosis (OP-ADSCs) in vitro by tissue explant culture and collagenase digestion, and to compare the efficacies of these two methods. METHODS: Adipose tissues were isolated from the inguen of C57BL/6 mice, from which OP-ADSCs were obtained by tissue explant culture and collagenase digestion respectively. Then, the cells were subjected to primary culture and subculture. The surface specific antigens of passage 3 cells were observed using flow cytometry, while the yields and proliferation abilities of passage 3 were compared at 7 days of culture. The adipogenic and osteogenic differentiation of OP-ADSCs at passage 4 was detected. RESULTS AND CONCLUSION: (1) The expression levels of CD34, CD146, Sca-1 in passage 3 cells were (15.22±1.85)%, (75.55±3.36)% and (83.48±4.22)% for the tissue explant culture and (13.46±2.21)%, (76.62±2.47)% and (84.84±3.56)% for the collagenase digestion method, respectively. (2) The cell yield from each milligram of adipose tissue by tissue explant culture was significantly higher than that by collagenase digestion (P < 0. 05). (3) The proliferation rate of cells in the tissue explant culture group was higher than that in the collagenase digestion group after 24 hours of culture. (4) The OP-ADSCs cultured by the two methods could differentiate into adipocytes and osteoblasts, and the lipid accumulation and the mineralized nodules showed no significant difference between the two groups (both P > 0.05). These experimental findings indicate that the tissue explant culture is more suitable for obtaining OP-ADSCs in vitro as compared with collagenase digestion, which contributes to provide adequate cell sources for studies on ADSCs treatment of osteoporotic fractures and bone defects. 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 Figures and Tables | References | Related Articles | Metrics

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Pro-apoptotic effect of a tumor suppressor gene WWOX on ovarian cancer stem cells and its mechanism Wang Yi-yi, Zhang Xiang-ning, Dong Hao, Gao Lei, Cui Xiao-rong, Wang Chun-yan, Ma Rui-lin 2017, 21 (29):  4660-4665.  doi: 10.3969/j.issn.2095-4344.2017.29.012 Abstract ( 330 )   PDF (1099KB) ( 211 )   Save BACKGROUND: WWOX, a tumor suppressor gene, can affect the growth of ovarian cancer stem cells; however, there is no report on whether its mechanism of action is related to Hedgehog signaling pathway. OBJECTIVE: To investigate the effect and mechanism of overexpression of WWOX on the apoptosis of ovarian cancer stem cells. METHODS: pcDNA3.1-WWOX (pcDNA3.1-WWOX group) and pcDNA4.0-WWOX (pcDNA4.0-WWOX group) were transferred into ovarian cancer stem cells, respectively; and meanwhile, pcDNA3.1 (pcDNA3.1 group) and pcDNA4.0 (pcDNA4.0 group) were transferred into the cells. A non-transfection group (only with Lipofectamine2000) was set up. After cultured 48 hours, the levels of WWOX in the pcDNA3.1-WWOX group and pcDNA4.0-WWOX group were detected using western blot assay, and the cell proliferation and apoptosis were detected using MTT assay and flow cytometry, respectively. Western blot assay was also used to detect the levels of Hedgehog signaling pathway associated proteins, SHH, PTCH1, Gli-1, SMO and apoptosis-related protein Cleaved Caspase-3 in the cells. Cyclopamine, Hedgehog signaling pathway inhibitor, was used in ovarian cancer stem cells without transfection (cyclopamine group) and after the transfection of WWOX overexpression vector (WWOX+cyclopamine group) followed by 48 hours of culture, and then MTT, flow cytometry and western blot detections were performed. RESULTS AND CONCLUSION: (1) The expression level of WWOX in the pcDNA4.0-WWOX group was significantly higher than that in the pcDNA3.1-WWOX group (t=27.84, P=0.00). The ovarian cancer stem cells which were transfected with pcDNA4.0-WWOX were used to overexpress WWOX in the late experiment. (2) Overexpression of WWOX could inhibit the proliferation of ovarian cancer stem cells and promote the apoptosis of ovarian cancer stem cells. (3) Overexpression of WWOX could inhibit the expression of Gli-1, PTCH1, SMO and SHH in ovarian cancer stem cells, and promote the expression of Cleaved Caspase-3. (4) Cyclopamine could inhibit the expression of SHH, PTCH1, Gli-1, SMO, and promote the expression of Cleaved Caspase-3. Cyclopamine had obvious inhibitory effect on Hedgehog signaling pathway. (5) Cyclopamine could enhance the apoptosis induced by overexpression of WWOX in ovarian cancer stem cells , and enhance the inhibition of proliferation of ovarian cancer stem cells induced by overexpression of WWOX. To conclude, WWOX effects on proliferation and apoptosis of ovarian cancer stem cells may be related to the inhibition of Hedgehog signaling pathway. 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 Figures and Tables | References | Related Articles | Metrics

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Biological characteristics and clinical observation of autologous nasal mucosa mesenchymal stem cells in the treatment of spinal cord injury Zhuo Yi, Duan Da, Ge Li-te, Yuan Ting, Liu Bo, Wu Pei, Wang Hao, Long Lang, Liu Zuo, He Xi-jing, Lu Ming 2017, 21 (29):  4666-4672.  doi: 10.3969/j.issn.2095-4344.2017.29.013 Abstract ( 399 )   PDF (1988KB) ( 221 )   Save BACKGROUND: Stem cells have been widely used in the treatment of spinal cord injury, but the clinical application is limited by immune rejection, difficulty in cell harvesting and purification. However, human nasal mucosa mesenchymal stem cells (hNM-MSCs) have no such problems, and its clinical application in the treatment of spinal cord injury has been not reported yet. OBJECTIVE: To observe the biological characteristics of autologous hNM-MSCs and its clinical efficacy in the treatment of advanced incomplete spinal cord injury. METHODS: NM-MSCs were isolated from the human nasal mucosa, cultured and identified in vitro. The ultrastructure of NM-MSCs was observed by transmission electron microscope and scanning electron microscope. Then the NM-MSCs were induced to differentiate into osteocytes, adipocytes, stem cell spheres, or neurons in vitro. Totally eight patients with incomplete spinal cord injury were enrolled and subjected to hNM-MSCs transplantation via lumbar puncture for 1-3 sessions of about 5×107 cells each, with an interval of 5-7 days, after the approval of the medical ethics committee. All the patients were followed up for 6 months. Preoperative and postoperative therapeutic effect evaluations were performed on the basis of American Spinal Injury Association (ASIA) scores. RESULTS AND CONCLUSION: Under light microscope, the NM-MSCs were mainly spindle-shaped, positive for STRO-1 and exhibited a radial arrangement. NM-MSCs highly expressed CD73, CD90 and CD105, but did not express CD34 and CD45, with the purity of over 97%. And lots of podgy microvilli were seen on the surface of NM-MSCs under the scanning electron microscope, and two kinds of cell morphologies were seen under the transmission electron microscope. Moreover, hNM-MSCs had the ability to differentiate into osteocytes, adipocytes, stem cell spheres and neurons. During the 6-month follow-up, seven patients achieved neurological function recovery to different extents except for one patient, and no side effects were found. It is concluded that hNM-MSCs can become the ideal seed cells for tissue-engineered cell repair. Autologous NM-MSCs transplantation for the treatment of spinal cord injury can achieve the ideal effect, with the value of clinical application. 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 Figures and Tables | References | Related Articles | Metrics

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Salvia miltiorrhiza injection combined with adipose-derived stem cell transplantation improves neurologic function of cerebral infarction rats Yang Peng, Gao Da-wei 2017, 21 (29):  4673-4678.  doi: 10.3969/j.issn.2095-4344.2017.29.014 Abstract ( 281 )   PDF (1194KB) ( 179 )   Save BACKGROUND: Both salvia miltiorrhiza injection and adipose-derived stem cells have evident neuroprotection against cerebral infarction in rats. OBJECTIVE: To observe the effect of salvia miltiorrhiza injection combined with adipose-derived stem cell transplantation on cerebral infarction in rats and the levels of cyclooxygenase-2 (COX-2) and beta-amyloid protein (Aβ) in the serum and brain tissues.  METHODS: A rat model of cerebral infarction was successfully established in 63 rats, and then the model rats were randomly divided into three groups (n=21 per group): model group (cerebral infarction group with 30 μL of PBS via the tail vein), stem cell group (transplantation of adipose-derived stem cells, 30 μL, 3×106/L, via the tail vein), salvia miltiorrhiza injection + adipose-derived stem cell transplantation(combined group with transplantation of adipose-derived stem cells, 30 μL, 3×106/L, via the tail vein and intraperitoneal injection of salvia miltiorrhiza injection, 0.5g/(kg•d) for consecutive 7 days). The modified neurological severity scores of each group were evaluated before and at 1, 2, 3, and 4 weeks after treatment. At 3 weeks after treatment, MTT assay was used to detect the serum levels of COX-2 and Aβ; 2,3,5-triphenyltetrazolium chloride staining was performed to detect infarction size; and RT-PCR and western blot assay were used to detect the mRNA and protein levels of COX-2 and Aβ in the brain tissues. RESULTS AND CONCLUSION: The modified neurological severity scores in the three groups were ranked as follows: combined group < stem cell group < model group, and there were significant differences between groups (P < 0.05). Compared with the model group, the levels of COX-2 and Aβ in the serum and brain tissues at protein and mRNA levels were significantly lower in the stem cell group and combined group; compared with the stem cell group (P < 0.05), the levels of COX-2 and Aβ were significantly lower in the combined group (P < 0.05). The infarct size was smallest in the combined group, followed by the stem cell group, and biggest in the model group (P < 0.05). To conclude, salvia miltiorrhiza injection combined with adipose-derived stem cell transplantation can improve the neurological function of rats after cerebral infarction, probably through reducing the levels of COX-2 and Aβ in the rat brain and serum. 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 Figures and Tables | References | Related Articles | Metrics

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Effect of human umbilical cord mesenchymal stem cells on immune reconstruction of acute lymphoblastic leukemia children undergoing allogeneic hematopoietic stem cell transplantation Xiang Jin-feng 2017, 21 (29):  4679-4684.  doi: 10.3969/j.issn.2095-4344.2017.29.015 Abstract ( 438 )   PDF (1197KB) ( 244 )   Save BACKGROUND: Human umbilical cord mesenchymal stem cells (hUC-MSCs) can secrete a variety of cytokines and growth factors to support hematopoiesis. They can change the hematopoietic microenvironment, and furthermore, regulate and improve the immune function of the organism. OBJECTIVE: To investigate the effect of hUC-MSCs on immune reconstitution and prognosis in children with acute lymphoblastic leukemia (ALL) after allogeneic hematopoietic stem cell transplantation (allo-HSCT).  METHODS: Sixty-four ALL children who had undergo allo-HSCT were enrolled and randomized into non-blood donor allo-HSCT group (n=32) and non-blood donor allo-HSCT+hUC-MSCs group (n=32). The therapeutic effect and complication were observed in the two groups. The changes of immunoglobulins and lymphocyte subpopulations in the two groups were compared before and 1, 3 and 6 months after transplantation.  RESULTS AND CONCLUSION: Compared with the allo-HSCT group, the time of granulocyte implantation was shorter (P < 0.05), and the incidence of chronic graft versus host disease and cytomegalovirus was lower in the allo-HSCT+ hUC-MSCs group. There were no significant differences in hematopoietic stem cell implantation rate, platelet remodeling rate, cumulative recurrence rate and 1-year survival rate between the two groups (P > 0.05). The levels of serum immunoglobulins IgA, IgG, IgM, IgE and the levels of T lymphocytes (CD4+, CD3+CD4+, CD4+CD8+) and B cells in the peripheral blood of allo-HSCT+hUC-MSCs group were significantly higher than those in the allo-HSCT group at 1, 3 and 6 months after transplantation (P < 0.05). In conclusion, the combined use of hUC-MSCs infusion and allo-HSCT can effectively reduce the incidence of chronic graft-versus-host disease, and contribute to granulocyte hematopoietic reconstitution and immune function reconstruction. 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 Figures and Tables | References | Related Articles | Metrics

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Effect of different doses of adipose-derived stem cells on early renal fibrosis Lv Chun-yan, Li Jin-hui, Liu Wei Wei, Chen Chang-jin, Wu Cheng, Zhao Zi-yi, Shen Dong-cheng 2017, 21 (29):  4685-4690.  doi: 10.3969/j.issn.2095-4344.2017.29.016 Abstract ( 437 )   PDF (5315KB) ( 235 )   Save BACKGROUND: Our preliminary findings have shown that stem cells have a certain effect on early formation of renal fibrosis and delay the occurrence of renal interstitial fibrosis. OBJECTIVE: To investigate the effect of exogenous adipose-derived stem cell transplantation on the formation of renal interstitial fibrosis in rats, and to explore the dose-effect relationship.  METHODS: Fifty Sprague-Dawley rats were randomly divided into five groups. Rats in sham operation group were injected with PBS; and those in the other four groups were ligated to establish the model of renal fibrosis. After successful modeling, the model group was injected with PBS, while low dose group, middle dose group and high dose group were injected with 1×107/L, 2×107/L, 3×107/L adipose-derived stem cell suspension (0.1 mL), respectively. Rats were killed 14 days after injection. Hematoxylin-eosin staining and Masson staining were used to observe the degree of renal tubular interstitial injury and the relative area of the renal interstitium. Expression of alpha smooth muscle actin and transforming growth factor beta1 was detected by immunohistochemistry.  RESULTS AND CONCLUSION: (1) Hematoxylin eosin staining: The model group showed typical glomerular and tubulointerstitial changes; the low, middle and high dose groups had a certain degree of glomerular and tubulointerstitial changes, but the severity of injury in these three groups, especially in the high dose group, was significantly milder than that in the model group. (2) Masson staining: In the model group, the renal interstitium was widened and the collagen fibers were deposited; in the low, middle and high dose groups, the degree of renal interstitial expansion was lower than that in the model group, and moreover, the degree of renal interstitial expansion in the middle and high dose groups was lower than that in the low dose group (P < 0.05). (3) Immunohistochemical staining: Compared with the sham operation group, the expression of alpha smooth muscle actin and transforming growth factor beta1 in the model group was higher than that in the sham operation group (P < 0.05). Compared with the model group, significantly decreased expression of alpha smooth muscle actin was found in the high dose group (P < 0.05) and significantly reduced transforming growth factor beta1 expression was observed in the high, middle, and low dose groups (P < 0.05), especially in the middle and high dose groups. To conclude, exogenous adipose-derived stem cell transplantation can delay the formation of early renal fibrosis in a dose-effect manner, which may be realized by changing the signal pathway state induced by transforming growth factor beta1. 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 Figures and Tables | References | Related Articles | Metrics

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Clinical monitoring of chromosome karyotype and fusion gene expression after allogeneic hematopoietic stem cell transplantation in chronic myelocytic leukemia Li Zheng-fa, Liu Wei, Du Yun-yun, Yang Tong-hua, Zhao Jie, Shi Ke-qian, Tang Xin-hua, Yang Yan-mei,Zhang Yin-hong, Lai Xun, Wen Yan, Lu Zhi-xiang 2017, 21 (29):  4691-4696.  doi: 10.3969/j.issn.2095-4344.2017.29.017 Abstract ( 368 )   PDF (4163KB) ( 224 )   Save BACKGROUND: It has been reported that 70% of patients with chronic myeloid leukemia (CML) are negative for cytogenetic and genetic markers within 1-5 months after allogeneic hematopoietic stem cell transplantation (allo-HSCT), but there are still some patients who have repeatedly varied outcomes in cytogenetic and genetic marker detection. Overall, the negative rate is up to 89.5% at 3-12 months after allo-HSCT. OBJECTIVE: To monitor the changes in cytogenetic and genetic marker expression and to explore the prognostic significance in CML patients undergoing allo-HSCT. METHODS: Seventeen CML patients who had undergone allo-HSCT were enrolled. Chromosome G banding pattern of the bone marrow from these patients were analyzed using short-term culture method and direct method at 30 days, 2, 3, 4, 6, 12, 24, 36, 48, 60, 72 months after allo-HSCT. Dual-color fluorescence in situ hybridization was used to detect bcr-abl fusion gene; bcr-abl expressions in primary bone marrow cells from CML patients were detected using RQ-PCR. RESULTS AND CONCLUSION: There were 8/17 cases of male patient/male donor and 7/17cases of male patient/female donor (compatriots). 46XX karyotype (women) was detected by multiple reexaminations after transplantation, and there was no Y chromosome or other aberration of chromosome karyotype in their karyotype. Among the 17 cases, 1 case of female patient/female donor (compatriots) and 1 case of female patient/male donor (unrelated) manifested 46 XY chromosome karyotype and bcr-abl positive at 1 month after transplantation; after 4 months, these two cases still maintained 46 XY chromosome karyotype but bcr-abl negative; after 4-96 months, the karyotype continued to remain as 46 XY, and bcr-abl (-). Among the 17 cases, 1 case of male patient/male donor of full-matched compatriot (brother) manifested that Ph chromosomal bcr-abl gene continuously expressed within 1-12 months after allo-HSCT; then the cases was given donor lymphocyte infusion, and the bcr-abl expression returned to be negative at 48 months after transplantation. To conclude, chromosomal karyotype analysis and bcr-abl fusion gene monitoring provide important reference value for subsequent treatment options and prognosis judgment for CML patients with allo-HSCT. 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 Figures and Tables | References | Related Articles | Metrics

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Influence of angiogenesis on neural stem cell proliferation in the subventricular zone after focal cerebral ischemia/reperfusion Li Zhi-ning, Han Wei, Rong Liang-qun, Gong Ai-ping, Lv You, Shan Jun-jun, Wei Xiu-e 2017, 21 (29):  4697-4702.  doi: 10.3969/j.issn.2095-4344.2017.29.018 Abstract ( 427 )   PDF (5093KB) ( 217 )   Save BACKGROUND: The angiogenesis may be related to the proliferation of neural stem cells, but there is still no unified view. OBJECTIVE: To observe the influence of angiogenesis on neural stem cell proliferation in the subventricular zone of rats after focal cerebral ischemia/reperfusion. METHODS: Male Sprague-Dawley rats were randomly divided into normal group, sham group, vascular endothelial growth factor (VEGF)+cerebral ischemia/reperfusion group, normal saline (NS)+cerebral ischemia/reperfusion group. The injection was done via the lateral cerebral ventricle. Then, each group was subdivided into four groups (1, 2, 7, 14 days after ischemia/reperfusion). Focal cerebral ischemia/reperfusion models were made by the thread method. After modeling, the corresponding intervention was given in each group. The expression changes of Nestin and vWF mRNA in the subventricular zone were detected in all groups by immunohistochemical staining and real-time PCR, respectively. RESULTS AND CONCLUSION: There was a certain increase in vWF and Nestin positive expression in the subventricular zone after cerebral ischemia/reperfusion. At 7 days after ischemia, the expression of vWF mRNA and Nestin reached the peak, indicating the proliferation of neural stem cells in the subventricular zone after cerebral ischemia/reperfusion is associated with the time of angiogenesis. In addition, the expression of vWF mRNA and Nestin was significantly higher in the VEGF+cerebral ischemia/reperfusion group than the other two groups, indicating angiogenesis could promote the proliferation of neural stem cells in the subventricular zone of rats after cerebral ischemia/reperfusion.  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 Figures and Tables | References | Related Articles | Metrics

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Transplantation of islet-like cells from human umbilical cord mesenchymal stem cells at different induced stages for the treatment of diabetic mellitus Shan Xia, Cui Xiao-lan, Shi Han, Shen Yi, Wang Jia, Zhang She-yi, Guo Bo, Wang Yi-zhong 2017, 21 (29):  4703-4708.  doi: 10.3969/j.issn.2095-4344.2017.29.019 Abstract ( 387 )   PDF (1137KB) ( 210 )   Save BACKGROUND: In previous studies, the human umbilical cord mesenchymal stem cells (hUC-MSCs) have been successfully differentiated into islet-like cells in vitro, and insulin expressions have been found. OBJECTIVE: To compare the therapeutic effects of different induced stages of islet-like cells differentiated from hUC-MSCs in a diabetic rat in vivo, so as to find the most suitable induced time in vitro and provide a theoretical basis for clinical treatment of diabetes mellitus. METHODS: Passage 3 hUC-MSCs were differentiated into islet-like cells after 7, 14, 21, 28 days of oriented induction. Eighty male healthy Wistar rats, clean grade, were used in the study. Except eight rats in normal control group, all the rats were injected with streptozotocin at a dose of 70 mg/kg to establish diabetic models. The rats at 10 days after successful modeling were randomly divided into model control group, non-induced group, 7-day induction group, 14-day induction group, 21-day induction group and 28-day induction group. Rats in the normal control group and model control group were given 2 mL of culture medium without any cells and rats in the other groups were implanted with corresponding cell suspension (2×106 cells) via tail vein for two sessions with an interval of 2 weeks. The blood glucose level, body mass and serum insulin level were detected during the treatment process. The rats were executed to observe the structure changes of each organ at 4 weeks after the second cell transplantation. RESULTS AND CONCLUSION: (1) Compared with the model control group, the body mass and the serum insulin level significantly increased and the blood glucose levels significantly decreased in all the transplantation groups (P < 0.05), and the therapeutic effect was best in the 28-day induction group. (2) Compared with the model control group and normal control group, the frozen sections in all the transplantation groups showed that the morphological structures of the liver and kidney were clear with no abnormal changes, such as necrosis and fibrosis, after transplantation. These experimental results show that it is relatively safe and effective to transplant the different induced stages of islet-like cells induced by hUC-MSCs in the treatment of diabetes mellitus, and the therapeutic effect of islet-like cells at 28 days of in vitro induction is most obvious. 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 Figures and Tables | References | Related Articles | Metrics

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Effect of Danshen injection with neural stem cells transplantation in rats with craniocerebral injury Liu Xiao, Wang Hu 2017, 21 (29):  4709-4715.  doi: 10.3969/j.issn.2095-4344.2017.29.020 Abstract ( 406 )   PDF (1254KB) ( 198 )   Save BACKGROUND: Danshen injection can dramatically enhance the proliferation and survival of neural stem cells (NSCs) after transplantation. OBJECTIVE: To investigate the effect of Danshen injection with NSCs transplantation in rats with craniocerebral injury. METHODS: Eighty-seven adult Sprague-Dawley rats were used to build severe traumatic brain injury models. After successfully modeling, the model rats were randomized into craniocerebral injury group (the subarachnoid injection of DMEM medium; n=20), Danshen injection group (the intraperitoneal injection of Danshen injection; n=20), and NSCs group (the subarachnoid injection of CM-Dil-labeled NSCs suspension; n=20) and combined group (intraperitoneal injection of Danshen injection plus the subarachnoid injection of CM-Dil-labeled NSCs suspension; n=20). The injection in each group was performed once a day for 3 consecutive days. The modified neurological severity scores were tested after 3 days, 1, 2, 3 and 4 weeks of the transplantation. The Morris water maze test was performed at 21-28 days of transplantation. NSCs survival and distribution were detected by fluorescence microscope and the pathologic changes of brain tissues were observed at 4 weeks after intervention. The expressions of synaptophysin and growth associated protein 43 at gene and protein levels were tested in the brain injury zone by RT-PCR and western blot assay respectively. RESULTS AND CONCLUSION: The modified neurological severity scores in the combined group were significantly lower than those in the other groups (P < 0.05). In the Morris water maze test, the average escape latencies in the combined group was significantly shortened in the combined group after 3-5 days of transplantation in comparison with the other three groups (P < 0.05 or P < 0.01); the frequency of passing through the platform and the percentage of the swimming distance at the target quadrant in the total swimming distance were significantly higher in the combined group than in the other groups (P < 0.05 or P < 0.01). The hematoxylin-eosin staining results showed the tissue voids in the Danshen injection and NSCs group were relatively smaller than those in the craniocerebral injury group, and the voids almost disappeared in the combined group. There were more CM-Dil positive cells in the combined group than the NSCs group. RT-PCR and western blot assay showed that the gene and protein expression levels of synaptophysin and growth associated protein 43 in the combined group were the highest followed by NSCs and Danshen injection groups, and the lowest expression levels were observed in the craniocerebral injury group. In summary, the Danshen injection combined with the NSCs transplantation can improve the neurological function of rats with severe craniocerebral injury, which might function by increasing the expression levels of synaptophysin and growth associated protein 43. 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 Figures and Tables | References | Related Articles | Metrics

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Therapeutic effect of autologous platelet rich plasma combined with bone marrow mesenchymal stem cell transplantation on long shaft fracture bone nonunion Zhang Song, Zhang Tao, Fu Gui-hong, Li Shun-hua, Zhou Xue-ru, Chen Long-kun 2017, 21 (29):  4716-4721.  doi: 10.3969/j.issn.2095-4344.2017.29.021 Abstract ( 287 )   PDF (1731KB) ( 248 )   Save BACKGROUND: Autologous platelet rich plasma and bone marrow mesenchymal stem cells have certain effects on bone repair, but there are rare reports on the clinical treatment of long shaft fracture bone nonunion using autologous platelet rich plasma combined with bone marrow mesenchymal stem cell transplantation. OBJECTIVE: To investigate the therapeutic effect of autologous platelet rich plasma combined with bone marrow mesenchymal stem cell transplantation on long shaft fracture bone nonunion. METHODS: Forty-seven patients with long shaft fracture bone nonunion were randomly divided into two groups: monotherapy group (n=22) and combination group (n=25). In the monotherapy group, autologous bone marrow mesenchymal stem cell transplantation was performed in the bone nonunion site. In the combination group, autologous platelet rich plasma combined with bone marrow mesenchymal stem cell transplantation was implemented in the bone nonunion site. Callus score, clinical healing time, local complications and limb function grade were recorded and compared between the two groups. RESULTS AND CONCLUSION: The healing properties and limb function in the combination group were significantly superior to those in the monotherapy group [healing time: (4.2±1.5) vs. (5.6±1.1) months, P < 0.05; healing rate: 92% vs. 86%, P < 0.05; callus score: 2.74±0.36 vs. 2.32±0.53, P < 0.05; limb function recovery rate: 77% vs. 84%, P < 0.05]. Complications like local skin redness or infection were not found in the two groups. In conclusion, both of the two methods can promote bone healing, but autologous platelet rich plasma combined with bone marrow mesenchymal stem cell transplantation has a better clinical effect on bone healing. 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 Figures and Tables | References | Related Articles | Metrics

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Culture and identification of endothelial progenitor cells from human adipose tissue with CD31 immunomagnetic beads Tang Wen-yan, Yang Yin-xiang, Wang Zhao-yan, Luan Zuo 2017, 21 (29):  4722-4727.  doi: 10.3969/j.issn.2095-4344.2017.29.022 Abstract ( 533 )   PDF (789KB) ( 546 )   Save BACKGROUND: Endothelial progenitor cells are precursor cells of mature endothelial cells, which can migrate to ischemic tissues and differentiate into mature endothelial cells, and then play an important role in vascular remodeling. Endothelial progenitor cells have wide application prospects in various ischemic diseases, but the biological characteristics and identification methods are still controversial. OBJECTIVE: To investigate the methods of isolation and culture of endothelial progenitor cells from the human adipose tissue and to identify their biological features, in order to provide a sufficient source of cells for ischemic diseases.  METHODS: Stromal vascular fraction cells were isolated from the human adipose tissue by enzymatic digestion, CD31+ cells were selected using immunomagnetic beads, and then cultured in endothelial basal medium-2 supplemented with the EGM-2-MV-SingleQuots. Endothelial progenitor cells were identified through detection of morphology, cell markers and cell functions.  RESULTS AND CONCLUSION: (1) CD31+ cells were selected by immunomagnetic beads and then cultured and amplified in vitro, which displayed typical cobblestone-like morphology, and they maintain their proliferative ability. (2) Flow cytometry results showed that the CD31+ cells expressed CD31 (98.84%), CD34 (97.21%), VEGRR2 (64.07%), CD146 (98.42%) and CD133 (2.55%), but hardly expressed CD45 (1.1%), a hematopoietic stem cell marker. (3) The CD31+ cells were also found to incept Dil-ac-LDL and exhibit lectin binding capability. Furthermore, a lumen-like structure was formed in Matrigel, which has the ability of angiogenesis in vitro. To conclude, these results suggest that it is feasible to isolate and culture endothelial progenitor cells from the human adipose tissue by enzymatic digestion combined with immunomagnetic bead sorting. 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 Figures and Tables | References | Related Articles | Metrics

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Mesenchymal stem cells homing to rat lung emphysema followed by differentiation into pulmonary vascular endothelial cells Zhao Xiao-jian, Lu Cai-ping, Chu Wei-wei, Zhang Ya-xiao, Zhang Bing, Zhen Qiang, Wang Ren-feng, Liu Jia-bao 2017, 21 (29):  4728-4733.  doi: 10.3969/j.issn.2095-4344.2017.29.02 Abstract ( 298 )   PDF (1244KB) ( 212 )   Save BACKGROUND: Mesenchymal stem cells have unique homing, immunoregulation and anti-inflammatory properties. After intravenous injection, mesenchymal stem cells can home to the damaged target organs and tissues, and function to repair damaged tissues. OBJECTIVE: To observe the pathological changes of lung tissues in rats with emphysema after mesenchymal stem cells transplantation. METHODS: Twenty-four female Wistar rats were randomly divided into experimental group, control group and healthy control group. In the first two groups, the model of pulmonary emphysema was established by the method of dropping porcine pancreatic elastase. BrdU-labeled mesenchymal stem cells were injected into the tail vein of the rats in the experimental group, and PBS was injected in the control group. After 14 days, the pathological changes of lung tissues were observed. Tumor necrosis factor-α level, alveolar wall apoptotic index, anti-CD34 and anti-BrdU immunohistochemical changes in the bronchoalveolar lavage fluid were detected. RESULTS AND CONCLUSION: Compared with the healthy control group, the tumor necrosis factor-α level and apoptotic index of alveolar wall cells increased (P < 0.01), and the relative areas of anti-Brdu and anti-CD34 decreased (P < 0.01) in the control group. Compared with the control group, the level of tumor necrosis factor-α and apoptotic index of alveolar wall cells decreased (P < 0.01), and the relative area of anti-Brdu and anti-CD34 increased (P < 0.01) in the experimental group. The histopathological findings showed that both the control group and the experimental group showed emphysema-like changes, but these changes were milder in the experimental group than the control group. To conclude, mesenchymal stem cells can inhibit inflammatory response and apoptosis in experimental emphysema, improve the pathological changes of the lung, and moreover, bone marrow mesenchymal stem cells can differentiate into lung vascular endothelial 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 Figures and Tables | References | Related Articles | Metrics

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Immunoregulatory mechanism of mesenchymal stem cells on the T and B cells in systemic lupus erythematosus Wang Ying-cui, Pan Xing-hua 2017, 21 (29):  4734-4741.  doi: 10.3969/j.issn.2095-4344.2017.29.024 Abstract ( 414 )   PDF (1123KB) ( 237 )   Save BACKGROUND: Studies have shown that the main functions of stem cells include secreting soluble factors, regulating immune balance, suppressing inflammatory response, directional differentiation into specific functional cells involved in tissue repair, promoting revascularization, improving blood circulation and anti-oxidative stress. Therefore, stem cells can be applied for treatment of autoimmune diseases, especially systemic lupus erythematosus. OBJECTIVE: To summarize the immunoregulatory mechanism of mesenchymal stem cells on T and B lymphocytes in systemic lupus erythematosus. METHODS: A computer-based online search of CNKI and PubMed from January 2006 to August 2016 was performed by the first author to search related articles with the keywords of “mesenchymal stem cell, systemic lupus erythematosus, immunoregulation” in Chinese and English, respectively. Articles related to the immunomodulatory effect of mesenchymal stem cells in the treatment of systemic lupus erythematosus were selected, and as for in the same field, literatures published lately in the authoritative journals were preferred. RESULTS AND CONCLUSION: A total of 207 literatures were initially selected, and finally 53 articles were involved in analysis according to the inclusion criteria. Mesenchymal stem cells have the potential of self-renewal and multi-directional differentiation, which are ideal seed cells for immune replacement therapy. Meanwhile, mesenchymal stem cells can treat autoimmune diseases radically because of the ability of immunoregulation. Currently, mesenchymal stem cells have been proved to be a safe and effective therapeutic measure in the treatment of systemic lupus erythematosus, which can avoid drug side effects and have a great clinical prospect. However, there are still numerous problems to be solved in clinic. 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 Figures and Tables | References | Related Articles | Metrics

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Replenishment of intestinal stem cells during the repair of radiation-induced intestinal injury Zhao Xin, Li Xiang-yang, Ji Wu 2017, 21 (29):  4742-4747.  doi: 10.3969/j.issn.2095-4344.2017.29.025 Abstract ( 505 )   PDF (954KB) ( 197 )   Save BACKGROUND: Radiation-induced intestinal injury causes a sharp reduction in the number of intestinal stem cells (ISCs) and their dysfunction. This brings great challenges in the repair of injured intestinal epithelium. Prompting restoration of the number and function of ISCs is a highlight of current research in the treatment of radiation-induced intestinal injury as well as in the repair of injured intestinal epithelium. OBJECTIVE: To review the latest progress in the pathway and mechanism of ISCs replenishment after radiation-induced intestinal injury. METHODS: The first author searched the CNKI, Wanfang and PubMed databases using the keywords of “intestinal stem cell, radiation-induced intestinal injury” in Chinese and English, respectively, to retrieve relevant articles published from January 2010 to June 2016. Then we generalized and summarized the results of these articles. RESULTS AND CONCLUSION: The fate of ISCs is regulated by various signal pathways, mainly including Wnt/β-catenin, BMP, Notch and EGF pathways. Early secretory progenitors (ESPs) are relatively quiescent during homeostasis, and insensitive to radiation injury; moreover, the ESPs can convert to ISCs under certain conditions. So, they are desired source of “reserve stem cells”. Stem cell niche plays an important role in reverse differentiation from ESPs to ISCs, but the exact regulatory mechanism remains largely unknown. Investigation on the above mechanism is of important scientific significance for deeply understanding the restoration process after radiation-induced intestinal injury and thus actively regulating intestinal injury repair. 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 Figures and Tables | References | Related Articles | Metrics

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A systematic review of stem cells in treatment of spinal cord injury and a network Meta-analysis of the therapeutic effects via different transplantation ways Liu Ying, Liu Chu-fan, Zhang Hui-ting, Shi Ye, Lan Shan, Tang Ling-ling, Ai Jin-wei, Pei Bin 2017, 21 (29):  4748-4756.  doi: 10.3969/j.issn.2095-4344.2017.29.026 Abstract ( 338 )   PDF (1816KB) ( 220 )   Save BACKGROUND: Stem cell transplantation has been used in the clinical treatment of spinal cord injury. However, the efficacy and safety are still controversial. Although there are many approaches for stem cell transplantation, which one is better is unclear as yet.  OBJECTIVE: To systematically evaluate the efficacy and safety of stem cell transplantation for spinal cord injury, and to compare the therapeutic difference in stem cell transplantation via different approaches.  METHODS: A computer-based online search was conducted in PubMed, The Cochrane Library (Issue 4, 2016), Embase, CNKI, VIP, CBM, and Wan-Fang databases up to May 13, 2016 to screen the relevant randomized clinical controlled trials of stem cells in the treatment of spinal cord injury. Two reviewers independently selected the studies, extracted information, and assessed the quality of included trials. Data extracted from eligible studies was pooled and meta-analyzed using Stata13.1 and Gemtc0.14.3 software. RESULTS AND CONCLUSION: A total of 10 randomized controlled trials involving 546 patients (294 in stem cells group and 252 in rehabilitation treatment group) were included. The results of meta-analysis showed that stem cell transplantation had an advantage over rehabilitation treatment in increasing American Spinal Cord Injury Association (ASIA) motor score, ASIA sensory score, Barthel Index, and decreasing the bladder residual urine volume. The incidence of low fever, abdominal distension, headache, lower limb numbness, and meningeal irritation was 14%, 7%, 7%, 8%, and 7%, respectively. Taking the rehabilitation treatment as a common reference, the results of the network meta-analysis showed that there were no significant differences in ASIA motor score, ASIA sensory score, Barthel Index, and incidence of complications among subarachnoid injection, intravenous injection, and intralesional injection. Compared with the rehabilitation treatment, only stem cell transplantation via subarachnoid injection had significant differences in ASIA motor score [MD=9.77, 95%CI (0.26, 21.46)], and ASIA sensory score [MD=25.79, 95%CI (10.07, 45.27)]. To conclude, the stem cells transplantation via subarachnoid injection is considered the most effective transplantation method. Due to the limitations of the included studies, more high-quality randomized controlled trials are required to verify the above conclusion. In addition, future studies should focus on the long-term efficacy and safety of stem cell transplantation in the treatment of spinal cord injury, and should investigate the clinical effects on spinal cord injury with different ASIA grades, types of stem cells, and transplantation time. 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 Figures and Tables | References | Related Articles | Metrics