结缔组织生长因子特异性小分子干扰RNA的筛选及其在抗肝纤维化中的作用

Inhibition effect of small interfering RNA targeting connective tissue growth factor on liver fibrosis in rats

目的:设计和合成针对抗肝纤维化关键基因结缔组织生长因子(connective tissue growthfactor,CTGF)的特异性小干扰RNA(siRNA),并筛选高效的CTGFsiRNA抗肝纤维化序列,探讨其通过尾静脉注射是否具有抗大鼠肝纤维化作用。方法:①筛选高效的CTGF siRNA序列。②干预肝纤维化模型大鼠。选取雄性SD大鼠30只,随机分成5组,分别为空白对照组尾静脉注射生理盐水8周;模型组腹腔注射40%CCl4(3 ml/kg)及尾静脉注射生理盐水,1次/3d,连续8周;预防组腹腔注射40%CCl4及尾静脉注射CTGF siRNA(0.1 mg/kg),1次/3 d,连续8周;2周治疗组腹腔注射40%CCl42周,后给予CTGF siRNA及CCl4 6周;4周治疗组腹腔注射40%CCl4 4周,后给予CTGFsiRNA及CCl4 4周。于最后一次CCl4注射3天后取血及组织标本,检测肝纤维化指标,应用Real-Time PCR及Western印迹法检测CTGF mRNA及蛋白质在大鼠肝组织表达,应用Masson染色检测肝组织纤维化。结果:与模型组(0.544±0.019)相比,预防组(0.105±0.003)及治疗组(0.190±0.006)大鼠肝组织CTGF mRNA和蛋白质表达显著下调(P<0.05),肝组织炎症和坏死及纤维化明显减轻,肝纤维化指标显著降低(P<0.05)。4周治疗组与模型组相比各指标降低,与预防组及2周治疗组相比各指标相对升高(P<0.05)。结论:成功筛选出高效的CTGF siRNA,且经尾静脉注射CTGF siRNA能显著抑制大鼠体内肝脏CTGF基因表达,并能有效防治大鼠肝纤维化,对于病程较长的肝纤维化也有一定的治疗作用,提示CTGF siRNA在抗肝纤维化治疗中有重要作用。

Objective: To design and synthesize small interfering RNA(siRNA) targeting connective tissue growth factor(CTGF) and to investigate its effect on liver fibrosis. Methods: The interference sequence of CTGF was designed and synthesized.Rat hepatic fibrosis model was induced by intraperitoneal injection of 40 % CCl4(3 ml/kg).Thirty male rats were randomly divided into 5 groups:in normal control and model groups rats received tail vein injection of normal saline every 3 days for 8 consecutive weeks;in preventive group rats received tail vein injection of CTGF siRNA(0.1 mg/kg) every 3 days for 8 weeks;in 2-w treatment group CTGF siRNA was given for 6 weeks starting from two weeks after CCl4 injection;in 4-w treatment group CTGF siRNA was given for 4 weeks starting 4 weeks after CCl4 injection.The serum and hepatic tissue samples were harvested 3 days after the last CCl4 injection.Hepatic fibrosis indices were measured.Expression of CTGF mRNA and protein in the liver was evaluated by RT-PCR and Western blot,respectively.Fibrosis in rat liver was analyzed by Masson staining. Results: Compared with model group(0.544 0.019),the expression of CTGF mRNA and protein in liver of both preventive(0.105±0.003)and 2-w treatment groups(0.190±0.006) were markedly down-regulated(P<0.05).Inflammation,necrosis and fibrosis in hepatic tissue were significantly attenuated.In addition,the serum ration of liver fibrosis indices was greatly reduced(P<0.05).Compared with preventive and 2-w treatment groups,the expression of CTGF mRNA and protein in liver in4 weeks of treatment group were up-regulated(P<0.05);inflammation,necrosis and fibrosis in hepatic tissue were relative increased;and the serum concentrations of liver fibrosis indices were relatively higher(P<0.05). Conclusion: The highly effective CTGF siRNA has been successfully synthesized,which can inhibit CTGF expression in liver,prevent hepatic fibrosis and its progress in rats.