The Study On MiR-210 Gene In Promoting The Formation Of Bone And Blood Vessel By Canine BMSCs In Vitro

Objective To explore the function of miR-210 gene in promoting the differentiation of BMSCs into bone and blood vessels with the gene transfection technology in vitro. To investigate whether targeted gene could promote the overexpressions of osteogenic and angiogenic factors, which could lay a foundation for the bone defect restoration in vivo in the future.Methods To culture the bone marrow mesenchymal stem cells in vitro,and then to detect the surface markers of BMSCs with flow cytometry The lentiviral vector carrying miR-210 or Green fluorescent protein(GFP) gene(Lenti-miR-210 or Lenti-Lac Z) was constructed and then transduced into the canine BMSCs. After transduced with targeted gene, the efficiency of Virus transduction is observed in inverted fluorescence microscope on day 3~5. The influence of the virus on the rate of proliferation of BMSCs was detected with the MTT method.After transduced with targeted gene,expressions of relative osteogenic and angiogenic factors were detected by RT-PCR and Western-blot on day 0,1,4,7,14 and 21. Alkaline phosphatase(ALP) and calcium nodules were detected by ALP staining and Alizarin red staining(ARS) after 21 days of transduction. SPSS(Statistical Product and Service Solutions) was used to analysis,all the data were described as mean±standard deviation, T test was used to compare the differences between groups and with P<0.05 as significant statistical differences.Results The BMSCs were observed in inverted phase contrast microscope, cells grew in the shape of spindle and spiral. Flow cytometry analysis suggested that surface antigens of marrow mesenchymal stem cells CD44 and CD90 were highly expressed,and these of hematopoietic stem cell CD34 and CD45 were negative expression,which could inferred that the cultured cells were BMSCs. when the multiplicity of infection(MOI) was ten,BMSCs have the highest transduction efficiency, the effect of virus on the proliferation of BMSCs was not obvious. On the fifth day after tranduced, transduction efficiency was the best(>90%). The BMSCs were successfully tranduced with miR-210 and GFP recombinant lentiviral vectors. After the targeted gene was transduced, expressions of VEGF and Runx2 at the m RNA and protein levels were significantly increased(P<0.05). Results of ALP and ARS staining showed that the targeted gene could induce the osteogenic differentiation of BMSCs more than Lenti-Lac Z and BMSCs.Conclusion Lentivirus could successfully transduced into the BMSCs.And the miR-210 gene could promote the overexpressions of osteogenic and angiogenic factors, which could lay a foundation for the relative study in vivo in the future.