【摘要】 目的:探讨miRNA-133b对骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMSCs)增殖及凋亡的影响,分析miRNA-133b影响BMSCs增殖的可能机制。方法:体外全骨髓贴壁法原代培养大鼠BMSCs,取生长良好的第2代细胞作为本实验的研究对象。利用siPORT NeoFX转染rno-miRNA-133b模拟物(mimic)、抑制剂(inhibitor)及其各自相对应的阴性对照(NC)。用带GFP荧光的miRNA和TaqManqRT-PCR验证转染效率,采用MTS法和Brdu细胞增殖比色法检测细胞增殖,TUNEL免疫荧光分析细胞凋亡。利用生物信息学方法预测miRNA-133b的靶点基因,并对其靶点基因进行基因功能分析。结果:①利用siPORT NeoFX转染大鼠BMSCs能有效实现细胞中miRNA-133b的过表达和抑制。②过表达miRNA-133b能明显促进BMSCs的增殖(P<0.05),而抑制miRNA-133b能明显减少BMSCs的增殖(P<0.05)。③miRNA-133b对BMSCs的凋亡无明显作用(P>0.05)。④生物信息学分析结果提示miRNA-133b的靶点中存在部分调节细胞增殖的基因位点。结论:miRNA-133b对BMSCs的增殖能力存在促进作用,其可能通过负调控多种增殖调节靶基因发挥作用。更多还原

【Abstract】 Objective: To investigate the effect of miRNA-133b on proliferation and apoptosis of bone marrow derived mesenchymal stem cells(BMSCs),and preliminary analyze its possible target and mechanism.Methods: Primary cultured BMSCs of rats were transfected with rno-miRNA-133b mimic and inhibitor by siPORT NeoFX to increase and reduce the activity of miRNA-133b,transfected with their corresponding negative control as control.The transfection rate were detected with qRT-PCR and fluorescence in the MSCs from FAM? dye-labeled miRNA.Cell proliferation was evaluated by MTS assay and colorimetric BrdU cell proliferation ELISA kit.Cell apoptosis was analyzed with TUNEL immunocytochemistry.Then the possible target genes of miRNA-133b were forecasted by bioinformatics tools,and the function of these genes was analyzed.Results: SiPORT NeoFX could effectively modify the miRNA-133b expression in BMSCs.Over-expression of miRNA-133b could significantly promote the proliferation of BMSCs(P <0.05),inhibition of miRNA-133b could inhibit the proliferation of BMSCs(P <0.05).Transfect miRNA-133b mimics and inhibitors into BMSCs had no significant effect on cell apoptosis.The possible target genes of miRNA-133b were forecasted by bioinformatics tools,and part of the target genes played a role in regulating cell proliferation.Conclusion: miRNA-133b promote the proliferation of BMSCs,which may regulate proliferation through negative regulation of multiple target genes.更多还原