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目的研究RNA干扰对系统性硬化病(SS)皮肤成纤维细胞结缔组织生长因子(CTGF)mRNA和蛋白表达水平的影响。方法设计4个针对CTGF的特异性小干扰RNA(siRNA)及一个非特异性siRNA。体外转录获得siRNA,将siRNA用6-羧基荧光素(FAM)标记后瞬时转染至原代培养的SS患者皮肤成纤维细胞;转染48h后,以RT-PCR及蛋白印迹法分别检测CTGFmRNA及蛋白量的变化。结果转染了4个特异性siRNA的成纤维细胞中CTGFmRNA水平均有不同程度下调(P<0.001),抑制效果由强至弱为,siRNA742>siRNA828>siRNA578>siRNA948。蛋白印迹则显示有3个siRNA能使CTGF蛋白表达水平不同程度下调(P<0.001),其中siRNA742的抑制效应最强。结论RNA干扰能显著抑制SS成纤维细胞CTGF的表达。
Objective To investigate the effect of RNA interference on the expression of connective tissue growth factor (CTGF) mRNA and protein in human dermal fibroblasts from patients with systemic sclerosis (SS). Methods Four small interfering RNA (siRNA) targeting CTGF and one nonspecific siRNA were designed. The siRNA was transcribed in vitro and siRNA was labeled with 6-carboxyfluorescein (FAM) and transiently transfected into primary cultured skin fibroblasts of SS patients. After 48 hours of transfection, CTGF mRNA and protein were detected by RT-PCR and Western blot, respectively Changes in protein content. Results The CTGF mRNA levels in four fibroblasts transfected with four siRNAs were all down-regulated to a certain extent (P <0.001). The inhibitory effect was stronger to the weaker. SiRNA742> siRNA828> siRNA578> siRNA948. Western blotting showed that 3 siRNAs could down-regulate the expression of CTGF protein (P <0.001), of which siRNA742 had the strongest inhibitory effect. Conclusion RNA interference can significantly inhibit the expression of CTGF in SS fibroblasts.