siRNA沉默Smad3对人KFB细胞增殖、凋亡及合成MMP3的影响

Effects of SiRNA-mediated Smad3 silence on proliferation,apoptosis,and MMP3 synthesis in human keloid fibroblasts

目的研究siRNA特异性沉默Smad3基因对人瘢痕疙瘩成纤维细胞(keloidfibroblast,KFB)增殖、凋亡及合成基质金属蛋白酶3(matrix metalloproteinase3,MMP3)的影响。方法设计合成3对Smad3特异性siRNA片段(Smad3-siRNA-Y1,Y2,Y3)和1对无关干扰片段,转染人KFB细胞,RT-PCR和Western blot方法检测Smad3-siRNA片段对Smad3基因的特异性沉默效果,MTT法检测其对细胞增殖的影响,流式细胞仪检测细胞周期及细胞凋亡的变化,免疫细胞化学法检测MMP-3表达量的变化。结果75nmol/L Smad3-siRNA-Y1处理KFB细胞48小时后可特异并有效地抑制Smad3基因的表达;抑制细胞增殖,使S期细胞减少,G0/G1期细胞增多;凋亡指数增加;细胞表达MMP-3增加。结论Smad3-siRNA可特异性抑制KFB细胞中Smad3基因表达,明显抑制细胞增殖,诱导细胞凋亡,并加快其合成MMP3。

Objective To investigate the effects of siRNA-induced specific silence of Smad3 on the proliferation,apoptosis,and matrix metalloproteinase 3 (MMP3) synthesis in human keloid fibroblasts (KFB).Methods Three Smad3 specific siRNA fragments (Smad3-siRNA-Y1,Y2,and Y3) and one irrelevant siRNA fragment were designed,synthesized,and transfected into human KFB cells. RT-PCR and Western blot were employed to validate the specific silencing effect of Smad3-siRNA on Smad3 gene. MTT assay was performed to detect KFB cell proli-feration,flow cytometry was used to analyze the cell cycle and apoptosis,and immunocytochemistry was conducted to measure the expression level of MMP3. Results Smad3 gene expression in KFB was inhibited specifically and efficiently by the treatment of Smad3-siRNA-Y1 for 48 h at a final concentration of 75 nmol/L; cell proliferation was significantly inhibited,with a reduction of S phase cells and a concomitant increase of G0/G1 phase cells; apoptotic index was increased,while the expression level of MMP-3 was elevated. Conclusion Smad3-siRNA could be employed to specifically inhibit the Smad3 gene expression in KFB cells,leading to significant inhibition of cell proliferation,obvious induction of cell apoptosis,and notable increase of MMP3 synthesis.