| Purpose:The lentivirus vector system with overexpression of mir-126 was successfully constructed and identified,and the optimal MOI was selected for transfection into BMSCs.BMSCs transfected with overexpressed mir-126 were implanted into SCI model rats to observe the survival,distribution and migration of mir-126 BMSCs in SD rats after transplantation.Method : 1.Bone marrow mesenchymal stem cells(BMSCs)were isolated and cultured from femur and tibia of SD rats to identify their stem cell capacity and phenotypic characteristics.2.The lentivirus vector system with overexpression of mir-126 was constructed and identified,and BMSCs were successfully transfected to select the best MOI.3.The proliferation multiple ability of BMSCs expressing mir-126 after transfection with overexpressed mir-126 was identified by fluorescence quantitative PCR,which verified that mir-126 BMSCs could be transferred stably and had no effect on the morphology,activity and stem cell characteristics of BMSCs.4.The modified Allen method was used to successfully create 50 spinal cord injury models in SD rats,which were divided into two groups: the over-expressed mir-126 BMSCs transplantation group and the blank vector GFP transplantation group,with a group of 25.5.BMSCs transfected with the lentivirus vector system of overexpressing mir-126 were transplanted into the acute spinal cord injury model of SD rats,and blank vector GFP was established in the SCI model of SD rats as the control group.At 12 h,1 day,3 days,1 week,and 2 weeks after cell transplantation,the operative field spinal cord sections of the two groups of rat models were sectioned,and the number,migration and survival time of BMSCs cells in the models were observed by fluorescence microscopy.Results: 1.Bone marrow mesenchymal stem cells(BMSCs)were successfully cultured and prepared by whole bone marrow adherent method,which can be differentiated into osteoblasts and adipocytes in vitro,and the phenotypes of CD34,CD45,CD29 and CD44 were identified.2.Lentivirus vector system with overexpression of mir-126 was successfully constructed and identified.BMSCs with stable overexpression of mir-126 were packaged by 293 T cells and screened.When MOI was 40,lentivirus transfection efficiency was high with overexpression of mir-126.After transfection,BMSCs mir-126 was highly expressed,and the expression level was about 6 times that of blank BMSCs.BMSCs were transfected with the lentiviral vector system expressing mir-126,and the expression of mir-126 BMSCs was stable and had no effect on morphology,activity and stem cell characteristics.3.50 SD rat models of spinal cord injury were divided into the over-expressed mir-126 BMSCs transplantation group and the blank vector GFP transplantation group,with 25 in each group.Spinal cord sections of rat models were taken at 5 different time points 12 h,1 day,3 days,1 week and 2 weeks after transplantation,and observed by immunofluorescence staining.Results of immunofluorescence staining: all the experimental data were processed with SPSS 19.0 software,and the values of each group were compared by one-way ANOVA test.The significance(Sig)of the overexpressed mir-126 BMSCs group and the GFP group at five different time points was greater than P,P=0.05,without statistical significance,and there was no significant difference between the two groups.These results suggested that mir-126 BMSCs could survive for a certain time after being transplanted into rats.Conclusion: This experiment adopts slow virus carrier system for expressing the miR-126 transfection BMSCs,can carry on the effective integration and expression in vitro,miR-126 BMSCs can stable to extend,and the cell morphology,proliferation and differentiation ability had no effect,the results show that overexpression of miR-126 after BMSCs transplantation in rats in vivo,cells can survive a certain time,for the next step in the regulatory mechanism in vivo study miR-126 in BMSCs experiment foundation. |
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