Smad3基因siRNA对TGF-β1双向调节成纤维细胞增殖的影响

Effect of small interfering RNA-mediated Smad3 gene silencing on transforming growth factor-β1-induced bi-directional effects on skin fibroblast proliferation

目的构建大鼠Smad3基因特异性siRNA沉默质粒,并探讨其在TGF-β1双向调节成纤维细胞增殖中的作用。方法构建大鼠Smad3基因沉默质粒,荧光纤维镜观察转染效率,定量PCR、免疫组化法检测Smad3基因和蛋白的变化,BrdU ELISA法和EMSA法分别检测转染siRNA及联合大小剂量TGF-β1刺激后细胞增殖和Smad3结合活性变化。结果沉默质粒转染效率为41.2%,瞬时转染后具有促增殖作用且与转染剂量成正比,并使其mRNA表达下调50%、蛋白表达明显降低,还可降低大小剂量TGF-β1双向调节细胞增殖和Smad3结合活性的变化程度。结论pSUPERSmad3沉默质粒构建成功,TGF-β1可通过调节Smad3来达到双向调节成纤维细胞增殖作用。

Objective To study the role of Smad3 in transforming growth factor-β1 （TGF-β1）-induced bi-directional effects on skin fibroblast proliferation.Methods The Smad3 small interfering （siRNA） plasmid was constructed using a pSUPER vector.The efficiency of cell transfection was detected by fluorescence microscopy,and the inhibitory effect of the plasmid was assessed by real-time quantitative RT-PCR and immunohistochemistry.The effect of the plasmid on the fibroblast proliferation and Smad3 binding activity was analyzed by BRDU ELISA and EMSA,respectively.Results The transfection efficiency of the plasmid into the cells was 41.2%.The Smad3 siRNA plasmid produced efficient and specific inhibition of the expression of Smad3,and promoted the cell proliferation in a dose-dependent manner and abrogated the bi-directional effect of TGF-β1 on the cell proliferation and Smad3 binding activity.Conclusion The siRNA targeting Smad3 gene can inhibit the protein expression and RNA transcription of Smad3,and TGF-β1 exerts bi-directional regulation on fibroblast proliferation by modulating Smad3 activity.