目的 探讨经腹腔注射抗结缔组织生长因子(CTGF)小分子干扰RNA(siRNA)是否能抑制大鼠体内肝脏CTGF基因表达及防治大鼠肝纤维化.方法 雄性SD大鼠30只,随机分成5组,分别为模型组腹腔注射40%CCl4 (3 mL/kg)及尾静脉注射生理盐水,1次/3 d,连续8周;预防组腹腔注射40%CCl4 及尾静脉注射抗CTGF siRNA(0.1 mg/kg),1次/3 d,连续8周;治疗组腹腔注射40%CCl4 2周,后给予抗CTGF siRNA及CCl4 6周;晚期治疗组腹腔注射40%CCl4 4周,后给予抗CTGF siRNA及CCl4 4周;空白对照组尾静脉注射生理盐水8周.于最后一次CCl4注射3 d后取血及组织标本,检测血清转氨酶、白蛋白、总胆红素及肝纤维化指标,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HE染色检测肝组织炎症及纤维化评分.结果 与模型组相比,预防组及治疗组大鼠肝组织CTGF mRNA及蛋白质表达显著下调(P<0.05),肝组织炎症、坏死及纤维化明显减轻,血清转氨基转移酶、肝纤维化指标显著降低.晚期治疗组与模型组相比大鼠肝组织CTGF mRNA及蛋白质表达下调(P<0.05),肝组织炎症、坏死及纤维化减轻,血清基转移酶、肝纤维化指标降低,与预防组及治疗组相比大鼠肝组织CTGF mRNA及蛋白质表达相对上调(P<0.05),肝组织炎症、坏死及纤维化程度相对增加,血清基转移酶、肝纤维化指标相对升高.结论 经尾静脉注射抗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,注射越早,越有效阻止肝纤维化的进展,且对于中晚期肝纤维化也有一定的治疗作用,提示抗CTGF siRNA将成为抗肝纤维化治疗的新靶点.%Objective To explore whether the intraperitoneal injection of small interfering RNA (siRNA) targeting connective tissue growth factor (CTGF) could inhibit CTGF expression in rats liver in vivo and prevent rats hepatic fibrosis. Methods Thirty male rats were randomly divided into five groups. Rats received intraperitoneally injection of 40％ CCl 4 (3 mE/kg) together with tail vein injection of saline every three days for 8 consecutive weeks were served as model group;CCl4 together with tail vein delivery of siRNA (0. 1 mg/kg) as preventive group;CCl4 for 2 weeks followed by CCl4 and CTGF siRNA for more than 6 weeks as curative group;CCl4 for 4 weeks followed by CCl4 and CTGF siRNA for more than 4 weeks as advanced curative group and only tail vein injection of saline for 8 weeks as control group. Blood sample and hepatic tissue from rats were harvested 3 days after the last CCl4 injection. Serum transaminase, albumin,total bilirubin and hepatic fibrosis indices were measured. The levels of CTGF mRNA and protein in rats liver was evaluated by RT-PCR and Western blot respectively. Inflammation and fibrosis in rats liver were analyzed on tissues sections with HE staining. Results Compared with model group, the expression of CTGF mRNA and protein in liver of both preventive and curative groups were markedly down-regulated(P＜0. 05). Inflammation , necrosis and fibrosis in hepatic tissue were significantly attenuated. In addition, the level of transaminases and liver fibrosis indices was greatly reduced. Compared with model group , the expression of CTGF mRNA and protein in liver in advanced curative group were down-regulated( P＜0. 05). Inflammation, necrosis and fibrosis in hepatic tissue were reduced. The level of transaminases and liver fibrosis indices were reduced. Compared with preventive and curative groups, the expression of CTGF mRNA and protein in liver in advanced curative group were up-regulated(P＜0.05). Inflammation , necrosis and fibrosis in hepatic tissue was increased. The level of transminases liver fibrosis indices was dlevated. Conclusion Tail vein delivery of CTGF siRNA could significantly inhibit CTGF expression in rat s liver in vivo,and effectively prevent rat s hepatic fibrosis. The earlier siRNA targeting CTGF was injected, the more preventive effect it had on the progress of liver fibrosis, especially on liver fibrosis in advanced stage, suggesting its potential in anti-fibrosis role in liver.
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