CTGF反义寡核苷酸对血管紧张素Ⅱ诱导的心肌成纤维细胞增殖、胶原合成的抑制效应

Inhibitory effects of antisense oligonucleotides targeting connective tissue growth factor mRNA on the proliferation and collagen synthesis of cardiac fibroblasts

目的 :研究结缔组织生长因子 (CTGF)反义寡核苷酸对血管紧张素Ⅱ诱导的心肌成纤维细胞增殖、胶原合成及CTGF表达的抑制作用。方法 :差速贴壁法分离新生SD大鼠心肌成纤维细胞 (CFs)。反义、正义、错义CTGF寡核苷酸分别经阳离子脂质体介导转染CFs,并与血管紧张素Ⅱ (AngⅡ ) 10 -6mol/L共培养 4 8h ,采用MTT法测CFs生长数目 ,羟脯氨酸法测胶原蛋白含量 ,RT -PCR法及Westernblotting法分别测CTGFmRNA及蛋白水平的表达。结果 :AngⅡ可以在mRNA及蛋白水平明显促进CFsCTGF的表达 (P <0 . 0 1) ,且呈浓度 -时间依赖性。CTGF反义链组的MTT反应A值、胶原蛋白含量、CTGFmRNA及蛋白水平的表达量均明显低于AngⅡ组 (P <0. 0 1) ,而CTGF正义链组和错义链组与AngⅡ组无显著差异 (P >0 .0 5 )。结论 :AngⅡ刺激下产生的CFsCTGFmRNA和蛋白水平的表达上调 ,可能是心肌纤维化传导通路中的关键环节 ,CTGF反义寡核苷酸可以序列特异性地阻断CTGF的介导 ,从而抑制AngⅡ对心肌成纤维细胞增殖、胶原合成及CTGF表达的诱导作用 ,进一步证实CTGF可能是拮抗心肌纤维化的特异性靶位。

AIM: To observe the effect of AngⅡ on the ex pression of connective tissue growth factor (CTGF) in cultured cardiac fibroblas t (CFs), the antisense oligonucleotides (ASOND) was used to investigate whether CTGF is necessary for the proliferation and collagen synthesis of CFs. METHODS: CFs of SD rats were isolated and cultured. The proliferation and collagen synthesis of CFs were a ssessed by MTT assay and measuring hydroxyproline, respectively. The expression of CTGF mRNA and protein were detected by reverse-transcription PCR (RT-PCR) and Western blotting, respectively. RESULTS: AngⅡ could significantly increase the expression of CT GF both at mRNA and protein level in a dose and time-dependent manner (P<0. 01). The MTT A value, the synthesis of collagen and the expression of CTGF i n AS OND group were all obviously lower than in AngⅡ group (P<0.01). There was no significantly difference among AngⅡ group, sense OND group and missense ON D group (P>0.05). CONCLUSION: It indicates that CTGF is involved in the process of myocardial fibrosis induced by AngⅡ, which may be a target for the treatment o f cardiac fibrosis.