舒筋接骨汤调控微小RNA-30a-5p/Sprouty1基因对骨髓间充质干细胞增殖和成骨分化能力的影响

Effects of Shujin Jiegu Decoction on the proliferation and osteogenic differentiation of bone marrow mesenchymal stem cells by regulating miR-30a-5p/Sprouty1 gene

目的 探讨舒筋接骨汤对人骨髓间充质干细胞(hMSCs)增殖和成骨分化能力的影响及其可能作用机制.方法 培养hMSCs,噻唑蓝(MTT)比色法检测不同浓度舒筋接骨汤生药和下调Sprouty1对细胞增殖的影响,实时荧光定量PCR(qRT?PCR)检测hMSCs中微小RNA?30a?5p(miR?30a?5p)表达水平,RT?qPCR和免疫印迹法检测不同分组细胞中Sprouty1及核心结合蛋白因子2(RUNX2)、成骨细胞特异性转录因子Osterix(OSX)mRNA和蛋白表达水平,双荧光素酶报告基因实验验证miR?30a?5p靶向调控Sprouty1的作用.结果 舒筋接骨汤呈浓度依赖性促进hMSCs增殖,1 mg/ml时促进细胞增殖效果最佳;与对照组比较,舒筋接骨汤处理可明显促进hMSCs中miR30a?5p及RUNX2、OSX mRNA和蛋白表达(P<0.05),抑制Sprouty1 mRNA和蛋白表达(P<0.05);双荧光素酶报告基因实验结果显示miR?30a?5p靶向负调控Sprouty1的表达;下调Sprouty1促进hMSCs增殖及细胞中OSX、RUNX2 mRNA和蛋白表达.结论 舒筋接骨汤可能通过调控miR?30a?5p/Sprouty1基因表达,促进hMSCs增殖和成骨分化.

Objective To investigate the effects of Shujin Jiegu Decoction on the proliferation and osteogenic differentiation of human bone marrow mesenchymal stem cells(hMSCs)and its possible mechanism.Methods hMSCs were cultured in vitro.Methylthiazole tetrazolium(MTT)colorimetric assay was used to detect the effects of Shujin Jiegu Decoction and Sprouty1 down?regulation at different levels on the proliferation of hMSCs.Real?time quantitative PCR(qRT?PCR)was used to detect the expression of micro RNA?30a?5p(miR?30a?5p)in hMSCs,and RT?qPCR and western blot were used to measure the mRNA and protein expression levels of Sprouty1,Runt?related transcription factor 2(RUNX2),and osteoblast?specific transcription factor Osterix(OSX)in different hMSCs groups.Dual luciferase reporter assay was used to validate the role of miR?30a?5p in targeted regulation of Sprouty1.Results Shujin Jiegu Decoction was shown to promote the proliferation of hMSCs in a concentration?dependent manner,exhibiting the most prominent effect at a level of 1 mg/ml.Compared with the control group,Shujin Jiegu Decoction could significantly promote the mRNA and protein expression of miR30a?5p,RUNX2 and OSX(P<0.05),and inhibit the mRNA and protein expression of Sprouty1(P<0.05),in hMSCs.The results of dual luciferase reporter gene assay showed that miR?30a?5p negatively target?regulated the expression of Sprouty1.Down?regulation of Sprouty1 promoted hMSCs proliferation and expression of OSX and RUNX2 mRNA and protein in hMSCs.Conclusion Shujin Jiegu Decoction may promote the proliferation and osteogenic differentiation of hMSCs by regulating the expression of miR?30a?5p/Sprouty1 gene.