siRNA沉默β-Catenin对肝星状细胞Ⅰ、Ⅲ型胶原表达的影响

The effect of interfering RNA targeting β-Catenin on the type Ⅰand Ⅲ collagen in hepatic stellate cells

目的研究siRNA干扰β-Catenin表达、阻断Wnt/β-Catenin信号通路对肝星状细胞(HSC)Ⅰ、Ⅲ型胶原表达的影响。方法将化学合成siRNAβ-Catenin以Lipofectamine包裹,转染HSC-T6细胞,设阴性对照和空白对照,抽提细胞总RNA及蛋白质,收集培养上清液,应用反转录-聚合酶链反应和Western blot法检测细胞β-Catenin表达;Western blot法检测细胞Ⅰ、Ⅲ型胶原表达情况;细胞免疫荧光法检测Ⅰ、Ⅲ型胶原表达与定位;酶联免疫吸附法检测培养上清液中Ⅰ、Ⅲ型胶原含量。结果转染siRNA的HSC-T6细胞β-Catenin基因及蛋白表达水平明显下调,Ⅰ、Ⅲ型胶原表达显著下调,免疫细胞化学提示转染siRNAβ-Catenin后的Ⅰ、Ⅲ型胶原表达均较对照组明显减弱;48h和72h时培养上清液中Ⅰ型胶原表达水平显著减少,分别比阴性对照下降(23.68±7.96)%和(38.42±6.58)%(F=18.26,P<0.01;F=26.38,P<0.01),Ⅲ型胶原含量于72h时降低(39.68±4.92)%(F=37.68,P<0.01)。结论 siRNAβ-Catenin能高效抑制HSC-T6细胞β-Catenin表达,阻断Wnt/β-Catenin信号通路,从而抑制HSC-T6细胞增殖,显著减少Ⅰ、Ⅲ型胶原的合成和分泌,具有预防及治疗肝纤维化的潜力。

Objective To investigate the effect of synthetic small interfering RNA（siRNA） targeting β-Catenin on the expression of type Ⅰ and Ⅲ collagen in hepatic stellate cells（HSCs）.Methods Synthetic siRNA targeting β-Catenin was transfected into HSC-T6 by lipofectamine package.Total RNA and protein of HSC-T6 cells were extracted.The expression of β-Catenin mRNA were detected by reverse transcription-polymerase chain reaction and the expression of β-Catenin protein was detected by Western blot.The expression and location of type Ⅰ and Ⅲ collagen were determined by using western blot and immunofluorescence.Enzyme linked immunosorbent assay was used to determine the content of type Ⅰ and Ⅲ collagen in the supernatants.Results The expression of β-Catenin mRNA and protein were markedly down-regulated in siRNA-transfected HSC-T6 cells.The expressions of typeⅠ,Ⅲ collagen were significantly reduced after siRNAβ-Catenin transfection when compared with the control group.The level of type I collagen in the supernatants of transfected cells was decreased than in the negative control at 48 and 72 hours.The level of type Ⅲ collagen decreased by（39.68±4.92）%（F=57.68,P0.01）at 72 hours.Conclusion Inhibition of β-Catenin by siRNA may potentially prevent and treat hepatic fibrosis thorugh inhibiting stellate cells proliferation and decreasing the synthesis and the secretions of type Ⅰ and Ⅲ collagen.