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目的研究小干扰RNA(siRNA)对胱抑素C(CysC)基因表达的抑制作用及对组织蛋白酶S(CatS)表达的影响。方法利用Ambion公司设计合成的以CysC为靶标的siRNA,通过脂质体转染人血管平滑肌细胞(VSMC),以未转染siRNA细胞和转染无关siRNA细胞为对照,采用RT-PCR和Western blot分别检测CysC在mRNA水平和蛋白水平的改变。选取抑制效率最高siRNA转染VSMC,以空白对照组、无关干扰组和CysC高表达组为对照,采用RT-PCR法和Western blot分别检测CatS在mRNA水平和蛋白水平的改变。结果转染24 h后,siRNA组CysC mRNA和蛋白表达均被有效抑制,与空白对照组相比,差异有统计学意义(P<0.01),其中,siRNA2的抑制效率最高。CatS的mRNA及蛋白表达水平在siRNA组均有明显升高,与其他三组比较,差异有统计学意义(P<0.01)。结论体外转录合成的siRNA可有效抑制人VSMC中CysC的表达,并对CatS基因表达有一定影响。
Objective To investigate the inhibitory effect of small interfering RNA (siRNA) on the gene expression of cystatin C (CysC) and its effect on the expression of cathepsin S (Cat S). Methods siRNA targeting CysC was designed and synthesized by Ambion and transfected into human vascular smooth muscle cells (VSMCs) by lipofectamine 2000. The untransfected siRNA and transfected siRNA were used as control. RT-PCR and Western blot CysC were detected in the mRNA level and protein level changes. The VSMCs with the highest inhibitory efficiency were selected and transfected into VSMCs. The control group, unrelated interfering group and CysC overexpression group were used as control. The mRNA and protein levels of CatS were detected by RT-PCR and Western blot respectively. Results The expression of CysC mRNA and protein in siRNA group was significantly inhibited after transfection for 24 h (P <0.01), and siRNA2 had the highest inhibitory efficiency. CatS mRNA and protein expression in the siRNA group were significantly increased, compared with the other three groups, the difference was statistically significant (P <0.01). Conclusion The siRNA synthesized in vitro can effectively inhibit the expression of CysC in human VSMCs and have some influence on the expression of CatS gene.