CTGF-siRNA对低氧诱导肺成纤维细胞胶原合成的影响

Effects of Small Interference RNA Targeting Connective Tissue Growth Factor on Hypoxia-induced Collagen Synthesis in Human Lung Fibroblasts

目的观察针对结缔组织生长因子(CTGF)的小干扰RNA(siRNA)对低氧诱导的人肺成纤维细胞胶原合成的影响。方法应用siRNA表达载体介导的RNA干扰(RNAi)技术,将CTGF-siRNA表达质粒稳定转染人肺成纤维细胞MRC-5,并将人肺成纤维细胞在低氧条件下(37℃,5%CO2,1%O2)培养1、2、4、6及12h。采用定量PCR法测定CTGF及Ⅰ型胶原基因表达,并应用Westernblot法检测CTGF及Ⅰ型胶原蛋白表达变化。结果低氧培养1h后,人肺成纤维细胞即可刺激诱导CTGFmRNA表达,其升高水平可持续至6h。而在CTGF-siRNA表达质粒稳定转染的MRC-5细胞内,低氧诱导的CTGF、Ⅰ型胶原基因及蛋白表达均降低(P<0.05)。结论针对CTGF的siRNA在体外可明显抑制低氧诱导的MRC-5肺成纤维细胞的胶原合成。

Objective To investigate the effects of small interference RNA （siRNA） targeting connective tissue growth factor（CTGF） on collagen synthesis induced by hypoxia in human lung fibroblasts. Methods CTGF- siRNA expression plasmid was stablely transfected into MRC-5. The transfected cells were cultured under hypoxia condition （37 ℃, 5%CO2, 1%O2） for 1, 2, 4, 6 and 12 hours respectively, The expression of CTGF and collagen type Ⅰ was assessed by real time quantitative PCR and Western blot. Results Expression of CTGF mRNA was upregulated as early as 1 hour in control scrambled-siRNA/MRC-5 cells after exposed to hypoxia condition, which remained the elevated level up to 6 hours. Compared with control cells, the expression of CTGF mRNA and protein induced by hypoxia was decreased significantly in CTGF-siRNA/MRC-5 cells. Moreover, the deposition of collagen type Ⅰ in CTGF-siRNA/MRC-5 cells was also declined. Conclusion The siRNA targeting CTGF gene could efficiently inhibit collagen deposition induced by hypoxia in human lung fibroblasts.