【摘要】 目的观察转化生长因子(TGF-β1)小干扰RNA(siRNA)对小鼠免疫性肝纤维化的阻断作用。方法设计3条针对TGF-β1不同靶位的短发夹RNA(shRNA),以及1条无关对照序列(HK),克隆至pGenesil-1质粒中,构建重组真核表达载体pGenesil-TGF-β1-m1、pGenesil-TGF-β1-m2、pGenesil-TGF-β1-m3和pGenesil-HK。30只雄性昆明小白鼠随机分为6组:正常组、模型组、无关序列对照组(pGenesil-HK)、序列1治疗组(pGenesil-TGF-β1-m1)、序列2治疗组(pGenesil-TGF-β1-m2)、序列3治疗组(pGenesil-TGF-β1-m3)。采用尾静脉注射刀豆蛋白A(Con A)制备免疫性肝纤维化动物模型。3个序列治疗组、无关序列对照组分别于第2、4、6周,在Con A注射后24 h内,尾静脉高压注射pGenesil-TGF-β1-m1、pGenesil-TGF-β1-m2、pGenesil-TGF-β1-m3和pGenesil-HK。第7周处死全部小鼠。常规生化方法测定肝组织羟脯氨酸(HYP)含量;应用半定量RT-PCR技术检测肝组织内TGF-β1、Smad3、Smad7、α-平滑肌肌动蛋白(α-SMA)mRNA的表达。结果治疗组小鼠HYP较模型组明显降低(P<0.01);RT-PCR显示治疗组肝脏内TGF-β1、Smad3及α-SMA mRNA表达较模型组明显降低(P<0.01),而Smad7 mRNA表达增强(P<0.01)。在3个治疗组中,pGenesil-TGF-β1-m1治疗组的疗效最佳。结论 TGF-β1 siRNA能有效抑制小鼠免疫性肝纤维化的发生,其机制可能是抑制肝内TGF-β1、Smad3及α-SMA表达,并促进Smad7表达,从而下调HSC的激活信号,减少胶原合成。更多还原

【Abstract】 Objective To investigate the inhibitory effect of siRNA targeted against transforming factorβ1 (TGF-β1) in mice with immune hepatic fibrosis induced by Concanavalin A(Con A).Methods Three short hairpin RNAs(shRNAs) targeting different positions of TGF-β1 were designed and cloned to a plasmid pGenesil-1 respectively to obtain three recombinant expression vectors(pGenesil-TGF-β1-m1,pGenesil-TGF-β1-m2 and pGenesil-TGF -β1-m3).Thirty male Kunming mice were randomly divided into 6 groups;normal group,model group,control group(pGenesil-HK) and three treatment groups(pGenesil-TGF-β1-m1,pGenesil-TGF-β1-m2 and pGenesil-TGF-β1 -m3).The immune hepatic fibrosis animal models were constructed by injecting Con A via the tail vein of mice that continued for 6 weeks.At the 2nd,4th and 6th weeks,three treatment groups were injected hydrodynamics based on transfection method via the tail vein within 24hrs after injection of Con A respectively.All of the mice were sacrificed at the 7th week.The levels of hydroxyproline(HYP) in liver tissue were examined by biochemistry method. The expressions of TGF-β1,Smad3,Smad7 andα-smooth muscle actin(α-SMA) mRNA in the liver were detected by semi quantitative RT-PCR.Results The levels of HYP of liver tissue in treatment groups were significantly lower than that in model group(P<0.01).The results of RT-PCR showed that the expression levels of TGF-β1, Smad3 andα-SMA mRNA were inhibited in treatment groups compared with model group(P<0.01),while the levels of Smad7 were increased(P<0.01).The inhibition effect of pGenesil-TGF-β1-m1 was the best among three treatment groups.Conclusion It is suggested that specific siRNA targeting TGF-β1 inhibite the liver fibrogenesis of immune hepatic fibrosis induced by Con A in mice.The anti fibrosis mechanisms of the siRNAs maybe associated with the down regulation of TGF-β1,smad3 and a-SMA expression and up regulation of Smad7 expression in liver tissue, which resulted in suppressing the activity of hepatic stellate cells.更多还原