siRNA干预CTGF和TIMP-1对大鼠肝星状细胞胶原分泌的影响

siRNA Interference of CTGF and TIMP-1 on Rat Hepatic Stellate Cell Collagen Secretion

目的研究小干扰RNA(small interfering RNA,siRNA)沉默结缔组织生长因子(connective tissue growthfactor,CTGF)和组织金属蛋白酶抑制剂-1(tissue inhibitor of matrix metalloproteinases,TIMP-1)对肝星状细胞(hepaticstellate cell,HSC)CTGF和TIMP-1基因表达以及对Ⅰ、Ⅲ型胶原分泌的影响。方法根据已筛选出的对CTGF和TIMP-1基因最有效的RNA干扰靶位,将化学合成siRNA CTGF和siRNA TIMP-1以脂质体LipofectamineTM2000介导,瞬时转染HSC-T6细胞,分别设siRNA CTGF组、siRNA TIMP-1组、siRNA CTGF和siRNA TIMP-1联合组、脂质体组及非特异性(negative control,NC)siRNA组,抽提转染24、48h细胞mRNA和蛋白,并收集细胞上清液。应用RT-PCR鉴定CTGF和TIMP-1mRNA的表达;Western blot检测其蛋白表达;ELISA法检测Ⅰ、Ⅲ型胶原的分泌。结果各干预组转染24、48h后分别与脂质体组和NC siRNA组比较,HSC-T6细胞CTGF及TIMP-1mRNA和蛋白表达均明显下调(均P<0.05),且干预组HSC-T6培养上清液中Ⅰ、Ⅲ型胶原含量明显减少(均P<0.05)。siRNA联合组分别与siRNA CTGF组和siRNA TIMP-1组比较,于转染48hsiRNA联合组较siRNA TIMP-1组抑制率高,细胞上清液中Ⅰ、Ⅲ型胶原量比siRNA TIMP-1组少,且差异具有统计学意义(均P<0.05);而与siRNA CTGF组比较,其CTGF mR-NA和蛋白抑制率增高,细胞上清液中Ⅰ、Ⅲ型胶原量较siRNA联合组减少,但差异无统计学意义(均P>0.05)。结论针对HSC-T6CTGF mRNA基因全长943位点和TIMP-1mRNA 304位点化学合成的siRNA,对靶基因mRNA和蛋白表达有较好的抑制效果,CTGF和TIMP-1沉默可显著减少细胞上清液中Ⅰ、Ⅲ型胶原含量;CTGF和TIMP-1两种基因同时沉默,有增强抗肝纤维化效果的可能。

Objective To investigate the effect of small interfering RNA（siRNA） targeting CTGF and TIMP 1 on type Ⅰ and Ⅲ collagen secretion in rat hepatic stellate cells. Methods The most effective siRNA targeting CTGF and TIMP-1 was transfected into HSC T6 cells by Lipofectamine package. Rat HSCs activated were divided into five groups： siRNA CTGF group,siRNA TIMP-1 group,siRNA CTGF combined with siRNA TIMP-1 group, Lipofectamine group and the negative con trol（NC） siRNA group. Total RNA and protein of the cells after transfection for 24 and 48 h were extracted, and the superna- tants were collected. The expression of CTGF and TIMP 1 was detected by using RT-PCR and Western blot. The contents of type Ⅰ and Ⅲ collagen in the supernatants were determined by using ELISA. Results As compared with NC siRNA and Lipo- fectamine groups,CTGF and TIMP-1 mRNA and protein expression levels were significantly decreased in the other treated groups after transfection for 24 and 48 h. The contents of type Ⅰ and Ⅲ collagen in the supernatants were significantly re- duced. The comparison of every treated group had statistically significant difference（P〈0.05）. There was significant difference in the suppression rate of TIMP-1 mRNA and protein expression and secretion of type Ⅰ and Ⅲ collagen in HSC-T6 cells be tween siRNA TIMP 1 group and siRNA CTGF combined with siRNA TIMP-1 group after transfection for 48 h（P〈0.05）, but there was no significant difference in the suppression rate of CTGF mRNA and protein expression between siRNA TIMP-1 group and siRNA CTGF combined with siRNA TIMP-1 group after transfection for 24 and 48 h（P〈0.05）. Conclusion siRNA chemically synthesized targeting CTGF gene and TIMP-1 gene at site of 943 and 304 excellently inhibits the expression of mRNA and protein in HSC-T6 cells, siRNA CTGF and siRNA TIMP-1 could significantly reduce secretion of type Ⅰ and Ⅲ collagen. The combination of siRNA CTGF and siRNA TIMP-1 may promote the effect of suppressing hepatic fibrosis.