RNA干扰选择性下调心肌成纤维细胞结缔组织生长因子

RNA Interference Selectivly Down-Regulated Connective Tissue Growth Factor by in Cardiac Fibroblast

目的研究RNA干扰技术能否选择性下调乳鼠心肌成纤维细胞结缔组织生长因子(CTGF)及其诱导的下游纤维化成分的表达。方法利用差速贴壁法分离培养乳鼠心肌成纤维细胞。用携带U6启动子和CTGF特异性短发夹RNA(shRNA)编码序列的质粒载体CTGF-shRNA,及含非特异性shRNA编码序列的对照质粒CTGF-con以lipofectamine ^(TM)2000为载体转染原代培养的乳鼠心肌成纤维细胞。加入含G418的DMEM培养液培养一月筛选后获得稳定转染的细胞。通过半定量RT-PCR检测细胞内转化生长因子(TGF-β_1),CTGF及其下游纤维化成分纤维连接蛋白(FN)的表达,Western-blot法检测细胞内CTGF及其下游纤维化成分纤维连接蛋白(FN)的表达。结果CTGF在心肌成纤维细胞中有基础表达,并在转化生长因子β_1(TGF-β_1)刺激下其含量增加,CTGF-shRNA使心肌成纤维细胞CTGF的mRNA和蛋白质的表达分别降低65%和78%,FN的mRNA和蛋白质的表达分别降低81%和63%,而TGF-β_1 mRNA的表达上调32%。转染对照质粒组较空白对照组与脂质体组CTGF,FN表达量差异无统计学意义。结论RNA干扰技术能选择性下调乳鼠心肌成纤维细胞CTGF及下游纤维化成分FN的表达,进一步为拮抗心肌纤维化的基因治疗提供了依据。

Objective To investigate whether the expression of connective tissue growth factor （CTGF） in neonatal rat cardiac fibroblasts can be down-regulated by RNA interference. Methods The neonatal rat cardiac fibroblasts were isolated and cultured. Then the cells were transfected by plasmids CTGF-shRNA which carrying an U6 promoter and a CTGF-specific shRNA-coding template sequence, or by plasmid CTGF-con which carrying an U6 promoter and nonspecific shRNA-coding sequence. After selection with G418 administration for one month, stable transfection were obtained in the cells. The mRNA expression of CTGF, TGF-β1 , and FN were measured by reverse transcription-polymerase chain reaction （RT-PCR）. Meanwhile, the protein expressions of CTGF, FN were detected by Western blot. Results Basal expression of CTGF was found in the control fibroblast cells. TGF-β1 induced remarkable the mRNA and protein expression of CTGF and FN. The plasmid CTGF-shRNA resulted in 65% and 78% decrease in CTGF mRNA and protein expression, while the mRNA and protein expression of FN were decreased by 81% and 63 %, associated with upregulation of TGF-β1 by 32 %. On the contrary, there was no significant difference in the cells transfected by the plasmid of CTGF-con. Conclusion These results indicate that RNAi can specifically knock down CTGF expression of neonatal rat cardiac fibroflast which may present a new strategy of gene therapy for myocardial fibrosis.