【摘要】 目的:研究miR-29b过表达对NIH/3T3细胞直接靶向作用及其细胞功能变化的影响。方法:将活化的NIH/3T3细胞,分为空白对照(Control)组、miR-29b mimics组、miR-Negative Control(miR-NC)组、TGF-β1组、TGF-β1+miR-29b mimics组、TGF-β1+miR-NC组。采用免疫荧光技术、RT-PCR及Western Blot检测miR-29b、COL1α1、COL3α1、TIMP-1、MMP-9、HSP47、α-SMA表达,流式细胞术检测细胞凋亡,CCK-8法检测细胞增殖。结果:miR-29bmimics组与miR-NC组和Control组比较,miR-29b表达上调,COL1α1及COL3α1mRNA及蛋白表达下调(P<0.05)。TGF-β1+miR-29b mimics组与TGF-β1组、TGF-β1+miR-NC组比较,miR-29b表达明显上调(P<0.01),COL1α1、COL3α1、TIMP-1、HSP47、α-SMA mRNA及蛋白均明显下降,MMP-9 mRNA及蛋白均明显升高(P<0.05)。TGF-β1+miR-29b mimics组与TGF-β1组、TGF-β1+miR-NC组比较早期凋亡明显增多,而细胞活力明显下降,与Control组比较差异有统计学意义(P<0.05)。结论:COL1α1及COL3α1是miR-29b的直接靶向调控分子,体外上调miR-29b表达可抑制TGF-β1致纤维化相关因子表达及细胞活力,并可促进细胞凋亡,为进一步研究体内肝纤维化奠定了基础。更多还原

【Abstract】 Objective: To study the overexpression of miR-29 b in NIH/3 T3 cell direct targeting and its cellular function changes. Methods: NIH/3 T3 cells were cultured and divided into six groups: the control group,miR-29 b mimics group,NC( negative control) group,TGF-β1 group,TGF-β1 + miR-29 b mimics group,TGF-β1 + miR-NC group. Adopting immunohistochemistry fluorescence technique,RT-PCR and Western Blot to detect the expression of miR-29 b,COL1α1,COL3α1,TIMP-1,MMP-9,HSP47 and α-SMA,while cell apoptosis and cell viability were detected by flow cytometry and CCK-8. Results: MiR-29 b expression increased in miR-29 b mimics group compared with MiRnegative control group and blank group,while COL1α1 and COL3α1 mRNA and protein expression decreased,and there is significant difference( P < 0. 05). MiR-29 b expression was significantly up-regulated in TGF-β1 + miR-29 b group( P < 0. 01),there is a significant difference compared with TGF-β1 group and TGF-β1 + miR-NC group,while COL1α1,COL3α1,TIMP-1,HSP47,α-SMA mRNA and protein were significantly decreased,MMP-9 mRNA and protein were significantly increased( P < 0. 05). Cells apoptosis were significantly increased in miR-29 b mimics group,while cell viability were significantly decreased,comparing to TGF-β1 and TGF-β1 + miR-NC group,with a significant difference compared with the control group( P < 0. 05). Conclusion: COL1α1 and COL3α1 are direct targets of regulatory molecules for miR-29 b,overexpression of miR-29 b can inhibits TGF-β1-induced fibrosis and cell viability,and can promote cell apoptosis which laid foundation for the further study of hepatic fibrosis in vivo.更多还原