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目的:研究肝星状细胞(HSC)中smad2特异性小干扰RNA(siRNA)对Ⅰ型胶原表达的抑制作用,探讨抗肝纤维化的基因治疗新方法。方法:设计合成靶向Smad2基因的siRNA,将筛选成功的siRNA瞬时转染入体外培养的肝星状细胞(HSC),并给予转化生长因子β(TGF-β)刺激,应用RT-PCR和Western blot技术检测对照组与实验组Ⅰ型胶原mRNA水平和蛋白水平表达差异,研究siRNA对Ⅰ型胶原表达的抑制作用。结果:siRNA能明显降低肝星状细胞中Smad2的RNA和蛋白的表达水平,证实筛选的siRNA有效,能特异性抑制Smad2的基因表达;TGF-β刺激肝星状细胞后,与对照组比较,siRNA转染组细胞外基质(ECM)成分Ⅰ型胶原的表达水平明显降低(P<0.05)。结论:siRNA能够抑制TGFβ对肝星状细胞的激活,阻断TGFβ-Smads传导通路,使Ⅰ型胶原分泌下调,有效抑制TGFβ诱导的肝纤维化。
Objective: To study the inhibitory effect of smad2-specific small interfering RNA (siRNA) on the expression of type Ⅰ collagen in hepatic stellate cells (HSC) and to explore a new method of gene therapy for anti-liver fibrosis. Methods: siRNAs targeting Smad2 gene were designed and synthesized. Transfected siRNAs were transiently transfected into hepatic stellate cells (HSCs) cultured in vitro and stimulated with transforming growth factor-β (TGF-β). RT-PCR and Western blot blot technique was used to detect the difference of type Ⅰ collagen mRNA expression and protein expression between control group and experimental group to study the inhibitory effect of siRNA on type Ⅰ collagen expression. Results: siRNA could significantly reduce the expression of Smad2 RNA and protein in hepatic stellate cells, and confirmed that the siRNA was effective and could specifically inhibit the expression of Smad2. After TGF-β stimulated hepatic stellate cells, compared with the control group, The expression of type I collagen in the extracellular matrix (ECM) of siRNA transfection group was significantly decreased (P <0.05). Conclusion: siRNA can inhibit the activation of hepatic stellate cells by TGFβ, block the TGFβ-Smads pathway, down-regulate the secretion of type Ⅰ collagen and effectively inhibit TGFβ-induced hepatic fibrosis.