miR-205促进hASCs细胞系向软骨细胞方向分化的作用及机制研究

Role and mechanism of miR-205 in promoting hASCs cell line differentiation into chondrocytes

目的探讨微小RNA(miR)-205通过调控核心结合因子α-1(Cbfα-1)表达促进人脂肪干细胞(hASCs)细胞系向软骨细胞方向分化的作用及机制。方法慢病毒转染法将Lenti-miR-205、Lenti-control慢病毒液分别转染至hASCs细胞,命名为miR-205过表达组、miR-NC组,另取未处理细胞为对照组;RT-qPCR法检测转染后miR-205基因表达量,MTT比色法检测细胞增殖;用甲苯胺蓝染色、免疫细胞染色及免疫荧光染色观察转染后第3代hASCs细胞诱导分化情况,RT-qPCR、Western blot法检测诱导分化2周后各组Cbfα-1、Smad3、TGF-β1、ColⅡmRNA和蛋白表达。结果转染后miR-205过表达组miR-205基因相对表达量高于miR-NC组和对照组(P<0.001)。miR-205过表达组转染后hASCs细胞培养3、7 d MTT试验吸光度(A)值高于对照组和miR-NC组(P<0.05)。诱导分化2周后,甲苯胺蓝染色显示,miR-205过表达组细胞染色呈强阳性深蓝染色,对照组和miR-NC组为微弱浅蓝色;免疫细胞化学染色显示,miR-205过表达组细胞及胞外基质强阳性棕黄染色,对照组和miR-NC组为弱阳性淡黄染色;免疫荧光染色显示,miR-205过表达组红色荧光强度较强,对照组和miR-NC组红色荧光强度较暗淡。miR-205过表达组诱导分化后Cbfα-1 mRNA和蛋白相对表达量低于对照组和miR-NC组,Smad3、TGF-β1、ColⅡmRNA和蛋白相对表达量高于对照组和miR-NC组(P<0.05)。结论miR-205过表达可促进hASCs细胞增殖、促进细胞向软骨细胞方向分化,可能通过抑制Cbfα-1、激活TGF-β1/Smad3信号通路发挥调控作用。

Objective To investigate the role and mechanism of microRNA(miR)-205 in promoting the differentiation of human adipose-derived stem cells(hASCs) cell lines to chondrocytes by regulating the expression of core-binding factor α-1(Cbfα-1).Methods Lenti-miR-205 and lenti-control lentivirus solution were transfected into the hASCs cells by lentivirus transfection method, which were named miR-205 overexpression group and miR-NC group. The untreated cells were taken as the control groups. The expression of miR-205 gene was detected by RT-qPCR and cell proliferation was detected by MTT colorimetry. The induced differentiation of the third generation hASCs cells after transfection was observed by toluidine blue staining, immunocytochemistry and immunofluorescence staining. The expressions of Cbfα-1, Smad3, TGF-β1, ColⅡ mRNA and protein were detected by RT-qPCR and Western blot.Results The relative expression leve of miR-205 gene in miR-205 overexpression group was higher than that in control group and miR-NC group(P<0.001). The absorbance(A) value of MTT test of the miR-205 overexpression group was higher than that of the control group and the miR-NC group(P<0.05). After 2 weeks of induction,the toluidine blue staining showed that the miR-205 overexpression group stained strongly positively in dark blue staining,while the control group and miR-NC group were weakly light blue. Immunocytochemical staining showed that the cells and extracellular matrix of miR-205 overexpression group were strongly positive brown-yellow staining,while those of the control group and miR-NC group were weakly positive yellowish staining.Immunofluorescence staining showed that the red fluorescence intensity of miR-205 overexpression group was stronger,and the red fluorescence intensity of control group and miR-NC group was dim. The toluidine blue staining in the control group and miR-NC group was weak light blue. The relative expression levels of Cbfα-1 mRNA and protein in the miR-205 overexpression group were lower than those in the control group and miR-NC group,and the relative expression levels of Smad3,TGF-β1,ColⅡ mRNA and protein in the miR-205 overexpression group were higher than those in the control group and miR-NC group( P < 0. 05). Conclusion MiR-205 overexpression can promote hASCs cell proliferation and differentiation towards chondrocytes,which may play a regulatory role by inhibiting Cbfα-1 and activating TGF-β1/Smad3 signaling pathway.