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| MENG Qingxi,WANG Weiheng,MENG Qingmei.[J].Chinese Journal of Spine and Spinal Cord,2018,(10):933-943. |
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| Received:September 23, 2017 Revised:July 26, 2018 |
| English Keywords:Spinal cord injury Stemcell therapy Bone mesenchymal stem cells Hypoxia Apoptosis Migration Microglia |
| Fund:国家自然科学基金资助项目（编号：81472071）；2016骨科围手术期血液管理项目（编号：2016-N-14-06） |
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| English Abstract: |
| 【Abstract】 Objectives: To investigate the effects of hypoxic preconditioning of bone marrow mesenchymal stem cells (BMSCs) on acute spinal cord injury(SCI) in rats and related mechanisms. Methods: BMSCs were isolated from 10 SD rats(55.6±4.2g) and cultured by modified whole bone marrow adherence methods, and then were transfected with green fluorescent protein(GFP) gene. In vitro, the proliferation ability of BMSCs was tested by using CCK-8 after treated by CoCl2 in different concentrations (0, 10, 50, 100, 200 and 300μM). The anti-apoptosis effect of serum deprivation after 0, 6, 12 and 24h culture migration was tested by using Transwell, and apoptosis was tested by using flow cytometry. In vivo experiment, vertical strike SCI model was used and hypoxic preconditioning BMSCs were injected subcutaneously, then the injected models were divided into sham operation group(group A), control group(group B), normal cell group(group C) and hypoxic preconditioning cell group(group D). The recovered neurological function was studied by BBB score before operation and 1, 3, 7, 10, 14, 21 and 28 days after operation. The pathological damage of spinal cord was evaluated by using HE staining 72h and 28d after SCI. Immunofluorescence staining was used to determine the transplanted cells viability and microglia activation. Results: In vitro experiments, the cck-8 results showed that the higher was the concentration of CoCl2, the greater was the effect on the proliferation capacity of BMSCs, and the 24h culture of 100μM CoCl2 could significantly reduce the proliferation ability of BMSCs(P<0.05) and increase the apoptosis of BMSCs(P<0.05). However, the number of cell migration of BMSCs at 6 and 12h after pretreatment significantly increased(P<0.05), and the apoptosis rate after serum deprivation culture for 24h was significantly reduced(P<0.05). In vivo experiments, compared with group B, the treatment of BMSCs could significantly improve BBB score of 14 days, 21 days and 28 days(P<0.05). BBB score of group D was significantly higher than that of group C at 21 and 28 days(P<0.05). HE staining results showed that BMSCs transplantation could significantly reduce cell death, bleeding and inflammatory cell infiltration at the spinal cord injury site. While compared with group C, the pathological injury of spinal cord in group D was more mild. After 72h of cell transplantation, green fluorescence cells were seen in both group C and group D, and the number of green fluorescence cells in group D(254.0±35.5) was significantly higher than that in group C(143.2±22.3, P<0.05). At 72h after SCI, iba-1 immunofluorescence staining showed that the number of microglia cells in group C(544.8±37.1) and group D(422.4±56.0) decreased significantly(P<0.05), and the ability of group D to inhibit the activation of microglia cells was higher(P<0.05), compared with group B(759.0±114.3). Conclusions: Hypoxic preconditioning manipulation of BMSCs is an effective way to improve the efficacy of cell transplantation in the treatment of SCI. |
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