摘要目的 观察慢病毒表达载体介导微小核糖核酸-34a(miRNA-34a)体外转染肝星状细胞-T6(HSC-T6)后对肝星状细胞增殖率及活化的影响,探讨miRNA-34a在肝纤维化中可能的作用.方法 体外培养HSC-T6细胞,分别对miRNA-34a重组慢病毒组和空白对照病毒组转染肝星状细胞,细胞计数试剂盒(CCK-8试剂盒)检测细胞增殖抑制率,实时荧光定量PCR(RT-PCR)、Western印迹法检测HSC-T6细胞转化生长因子-β1 (TGF-β1)、a-平滑肌肌动蛋白(α-SMA)和基质金属蛋白酶3 (MMP3) mRNA及蛋白表达.组间差异采用单因素方差分析,方差齐性者两两比较采用LSD法,方差不齐者采用Dunnett T3检验,相关性检验采用Pearson直线相关分析.结果 转染效率为80％；与空白对照病毒组相比,转染后RT-PCR显示,miRNA-34a重组慢病毒组miRNA-34a mRNA的含量是3.6,其表达量明显升高；miRNA-34a重组慢病毒组肝星状细胞增殖率明显降低(24、48及72 h的F值分别为9.048、168.070、812.390,均P＜0.05)；TGF-β1和α-SMA mRNA的表达量均降低,相对于空白对照病毒组的含量分别为0.28和0.13；TGF-β1和α-SMA蛋白表达量均降低(F=64.444、90.577,均P＜0.05)；而MMP3表达量明显升高(F=374.584,P＜0.05).结论miRNA-34a能抑制肝星状细胞增殖和活化,有望在治疗肝纤维化中发挥作用.

abstractsObjective To observe the influence of puromycin lentiviral vector (pLV)-miRNA34a transfection on proliferation and activation of rat hepatic stellate cells,and to investigate the mechanism of miRNA-34a on liver fibrosis in rat.Methods Hepatic stellate cells-T6 (HSC-T6) were transfected with pLV-EGFP or pLV-miRNA-34a,respectively.Cell proliferation was measured by C0038 cell counting kit-8 (CCK-8).The level of miRNA 34a,transforming growth factor (TGF)-β1,α-smooth muscle actin (α-SMA) and matrix metalloproteinasex3 (MMP3) expression were detected by real-time fluorescent quantitative-polymerase chain reaction (RT-PCR) and Western-blot.The comparison of means among groups was done by univariate ANOVA.Results The transfection efficiency was 80％.After hepatic stellate cells transfected with pLV-miRNA-34a,the expression of miRNA-34a mRNA was significantly increased compaired with the control group,and the content of miRNA-34a mRNA relative to the control group was 3.6.Cell proliferation of pLV-enhanced green fluorescent protein (EGFP)-miRNA-34a tranfected cells was lower than the control group (the value of F was 9.048,168.070,812.390 at 24,48,72 h respectively,all P＜0.05).After hepatic stellate cells were infected with pLV-EGEP-miRNA 34a,the expressions of TGF-β1 and α-SMA mRNA were lower than the control group,and the content of TGF-β1 and α SMA was 0.28 and 0.13 respectively.The expression of TGF-β1 and α-SMA proteins were significantly decreased (F=64.444 and 90.577,respectively； both P＜0.05),and the expression of MMP3 was significantly increased (F=374.584,P＜0.05).Conclusion miRNA-34a significantly inhibits the proliferation and activation of hepatic stellate cells,providing potential therapeutic implication for hepatic fibrosis.