RNA干扰对系统性硬化病皮肤成纤维细胞结缔组织生长因子表达的影响

The effect of RNA interference on the expression of CTGF in skin fibroblasts of systemic sclerosis

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 study the effect of RNA interference on the expression of CTGF in skin fibroblasts of systemic sclerosis （SS）. Methods Four CTGF specific siRNAs and a negative control siRNA were designed and then synthesized by in vitro transcription, siRNAs labeled with carboxyfluorescein-6-succimidyl ester （FAM） were transiently transfected into SS skin fibroblasts. Forty-eight hours after the fibroblasts were treated with siRNAs, the mRNA and protein expression of CTGF was detected by semiquantitatire RT-PCR and Western blot analysis, respectively. Results The mRNA and protein expression of CTGF in fibroblasts was significantly down-regulated by 4 and 3 CTGF specific siRNAs （both P 〈 0.001 ）, respectively, at different levels. Conclusion RNA interference could effectively suppress the expression of CTGF in SS skin fibroblasts.