过表达miR-378影响羊骨髓间充质干细胞成血管向分化

Overexpression of miR-378 affects vascular differentiation of sheep bone marrow mesenchymal stem cells

目的:研究过表达miR-378慢病毒对羊的骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSCs)成血管向分化的影响。方法:对羊BMSCs进行分离、培养及鉴定,进行过表达miR-378的慢病毒转染,细胞分为3组:过表达慢病毒转染BMSCs组(LV-miR-378-BMSCs组)、空载慢病毒转染BMSCs组(LV-BMSCs组),未转染组(空白组)。转染72 h后,流式细胞术与共聚焦显微镜检测转染效率。CCK-8实验进行细胞的毒性检测。细胞划痕实验检测miR-378对细胞迁移的影响。RT-qPCR检测成骨成血管相关基因mRNA的表达变化。结果:流式细胞仪检测慢病毒转染效率,LV-miR-378-BMSCs组、LV-BMSCs组、空白组转染率为86.4%、85.6%、0,两转染组与空白组比较明显增高,差异有统计学意义(P<0.05),而两病毒转染组比较差异无统计学意义(P>0.05)。CCK-8与细胞迁移实验显示,过表达miR-378可在一定程度上增强羊BMSCs的增殖能力与迁移能力(P<0.05)。RT-qPCR显示过表达miR-378可以显著提高miR-378、VEGF、PDGF、Ang-1、HIF-1α、BMP2的mRNA表达(P<0.001)。结论:过表达miR-378基因可以促进羊BMSCs成血管向分化。

Objective:To investigate the effect of miR-378 lentivirus expression on the vascular differentiation of bone marrow mesenchymal stem cells(BMSCs)from sheep.Methods:BMSCs from sheep were isolated,cultured,and identified,and transfected with miR-378 overexpression lentivirus.The cells were divided into three groups:overexpressing lentivirus transfected BMSCs group(LV-miR-378BMSCs group),empty lentivirus transfected BMSCs group(LV-BMSCs group),and non transfected group(blank group).After 72 hours of transfection,the transfection efficiency was detected by flow cytometry and confocal microscopy.CCK-8 experiment was conducted to detect cell toxicity.Cell scratch assay was used to detect the effect of miR-378 on cell migration.RT-qPCR was used to detect the expression changes of osteogenic vascular related genes mRNA.Results:Flow cytometry was used to detect the transfection efficiency of lentivirus.The transfection rates of LV-miR-378-BMSCs group,LV-BMSCs group,and blank group were 86.4%,85.6%,and 0,respectively.The transfection efficiency of the two transfection groups was significantly higher than that of the blank group,and the difference was statistically significant(P<0.05),while there was no statistically significant difference between the two virus transfection groups(P>0.05).CCK-8 and cell migration experiments showed that overexpression of miR-378 can enhance the proliferative and migratory abilities of sheep BMSCs to a certain extent(P<0.05).RT-qPCR experiments showed that overexpression of miR-378 significantly increased the mRNA expression of miR-378,VEGF,PDGF,Ang-1,HIF-1α,and BMP2(P<0.001).Conclusion:Overexpression of miR-378 gene can promote the angiogenic differentiation of sheep BMSCs.