微小核糖核酸-34a对肝星状细胞增殖及活化的影响  

Influence of puromycin lentiviral vector-miRNA-34a transfection on proliferation and activation of hepatic stellate cells

作　　者：宋梅[2] 王玉群[2] 曹肃婷[2] 林镯[2] 许烂漫[2] 施可庆[2] 王雅琴[2] 陈永平[2] 陈达之 

机构地区：[1]温州医学院仁济学院,325000 [2]温州医学院附属第一医院感染内科

出　　处：《中华传染病杂志》2013年第2期66-70,共5页Chinese Journal of Infectious Diseases

基　　金：浙江省大学生科技创新资助项目（新苗计划2012R413008）;艾滋病和病毒性肝炎等重大传染病防治资助项目（2013ZX10005002-001-008）;重型乙型肝炎（肝衰竭）临床治疗新方案的研究资助项目（2012ZX10002004）

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一34a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）、α-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DunnettT3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-34amRNA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72h的F值分别为9．048、168．070、812．390，均P〈0．05）；TGF-β1和a-SMAmRNA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。Objective To observe the influence of puromycin lentiviral vector （pLV）-miRNA- 34a 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, a-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 ofmiRNA-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 a 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.

关 键 词：微小核糖核酸 星形细胞 肝硬化 转化生长因子Β1 肌动蛋白类 基质金属蛋白酶3 

分 类 号：R285.5[医药卫生—中药学]