CTGF基因沉默对肺成纤维细胞增殖及表型转化的影响

近期研究表明,结缔组织生长因子(Connective tissue growth factor,CTGF)参与了肺成纤维细胞表型转化,与肺纤维化的形成关系密切.因此,本研究应用siRNA表达载体介导的RNA干扰(RNA interference,RNAi)技术,将CTGF-siRNA表达质粒稳定转染人肺成纤维细胞MRC-5,并通过定量PCR、Western-blot、细胞生长曲线及群体倍增时间、免疫细胞化学测定等方法检测基因表达、细胞增殖及表型转化等变化.结果显示,与非特异对照Scrambled-siRNA质粒稳定转染的MRC-5细胞或未转染MRC-5细胞相比,CTGF-siRNA表达质粒稳定转染的MRC-5细胞的CTGF表达明显降低;细胞增殖能力明显降低,群体倍增时间延长,由24.63或25.05 h延长至31.14 h(P＜0.05);TGFβ1刺激24 h后,细胞内α-平滑肌肌动蛋白α-smooth muscle actin,α-SMA)、Ⅰ型胶原及纤维连接蛋白(fibronectin,FN)的表达均明显下降.本研究提示,通过RNA干扰使CTGF基因沉默后,在体外可明显抑制MRC-5肺成纤维细胞的增殖、表型转化及细胞外基质的合成,可为基因治疗肺纤维化提供实验依据.

Effects of CTGF Gene Silencing on the Proliferation and Myofibroblast Differentiation of Human Lung Fibroblasts

Connective tissue growth factor (CTGF) is involved in the differentiation of lung fibroblast into myofibroblast and is considered as an important mediator in the pathogenesis of pulmonary fibrosis. In the present study, a CTGF small interference RNA (siRNA) expressing plasmid (CTGF-siRNA) was constructed and stably transfected into human lung fibroblast cell line, MRC-5. Stable clones with CTGF gene silencing (CTGF-siRNA/MRC-5) were successfully established by G418 screening and further confirmed by real-time quantitative PCR and Western blot. Cell proliferation was investigated by growth curve analysis, and cell doubling time of the CTGF-siRNA/MRC-5 cells was markedly longer than that of the control cells (P < 0.05). Compared with control cells, the expression of alpha-smooth muscle actin (alpha-SMA), the marker of myofibroblast differentiation, was decreased in CTGF-siRNA/MRC-5 cells. Moreover, the deposition of extracellular matrix (ECM) proteins (such as collagen type I and fibronectin) in CTGF-siRNA/MRC-5 cells was also declined. Our data suggest that CTGF may play an important role in the differentiation of lung fibroblast into myofibroblast, and that siRNA targeting CTGF gene might provide a new strategy for gene therapy of pulmonary fibrosis.