抗结缔组织生长因子小分子干扰RNA防治大鼠肝纤维化的研究

Research of small interfering RNA targeting connective tissue growth factor in prevention and therapy of rats liver fibrosis

目的探讨抗结缔组织生长因子（CTGF）小分子干扰RNA（siRNA）是否能抑制大鼠肝脏CTGF基因表达及防治大鼠肝纤维化。方法雄性sD大鼠30只，按数字表法随机分成5组，每组6只。模型组腹腔注射40％四氯化碳（CCI4）及尾静脉注射生理盐水，1次／3d，共8周；预防组腹腔注射40％CCI4及尾静脉注射抗CTGF siRNA，1次／3d，8周；早期治疗组腹腔注射40％CCI4 2周，后给予抗CTGF siRNA及CCI4 6周；晚期治疗组腹腔注射40％CCI4 4周后，给予抗CTGF siRNA及CCI4 4周；空白对照组尾静脉注射生理盐水8周。操作结束3d后取血及肝组织样本，检测大鼠肝功能及肝纤维化指标，应用实时PCR及Western印迹法检测CTGF mRNA及蛋白质在大鼠肝组织的表达，应用苏木精．伊红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、早期治疗组及晚期治疗组大鼠肝功能指标（ALT、AST、白蛋白和总胆红素）、肝纤维化指标（透明质酸、层黏连蛋白、人Ⅲ型前胶原和Ⅳ型胶原）、肝组织CTGF mRNA及蛋白质表达均显著下调（F=8．946～171．079，P〈0．05）；肝组织炎症、坏死及纤维化也明显减轻。与预防组及治疗组相比，晚期治疗组大鼠血清转氨酶、肝纤维化指标、肝组织CTGF mRNA及蛋白质表达上调（F=9．075～1526．644，P〈0．05）；HE染色肝组织炎症、坏死及纤维化程度增加。结论经尾静脉注射抗CTGF siRNA能显著抑制大鼠肝脏CTGF基因表达，并能有效防治大鼠肝纤维化，注射越早，越能有效阻止肝纤维化进展，对于中晚期肝纤维化也有一定的治疗作用，提示抗CTGF siRNA可成为抗肝纤维化治疗的新靶点。

Objective To explore whether small interfering RNA （siRNA） targeting connective tissue growth factor （CTGF） can inhibit CTGF gene expression and prevent and treat hepatic fibrosis occurrence. Methods 30 male rats were divided into 5 groups randomly, and 6 rats in each group. Rats received intraperitoneally injection of 40% CCI4 together with tail vein injection of saline every 3 days for 8 weeks were served as model group; CCI4 together with tail vein delivery of siRNA as preventive group; CCI4 for 2 weeks followed by CCI4 and CTGF siRNA for 6 weeks as early treatment group;CCI4 for 4 weeks followed by CCI4 and CI＇GF siRNA for 4 weeks as advanced treatment group, and only tail vein injection of saline as control group. 3 days after the last CCI4 injection, the serum and hepatic tissue from rats were harvested. Sennn transaminase, albumin, total bilirubin and hepatic fibrosis indices were measured. Expression of CTGF mRNA and protein in rats liver were evaluated by RT-PCR and Western blot, respectively. Inflammation and fibrosis in rats liver were analyzed by hematoxylin and eosin（HE）. Results Compared with the model group, the liver function （ALT,AST, albumin, TBil）, fibrosis in hepatic tissue（hyaluronic acid, laminin, human procollagen type Ⅲ, Ⅳ collagen） and the expression of CTGF mRNA and protein in preventive, early treatment and advanced treatment groups were markedly down-regulated（ F = 8.946-171.079, P 〈 0.05）; inflammation, necrosis and fibrosis in hepatic tissue were also relative down-regulated. Compared with preventive and early treatment groups, the expression of CTGF mRNA and protein in liver in advanced treatment group were up-regulated（ F = 9.075-1526.644, P 〈 0.05）. Inflammation, necrosis and fibrosis in hepatic tissue increased. Conclusions Tail vein delivery of CTGF siRNA can significantly inhibit CTGF expression in rats liver, and prevent rats hepatic fibrosis effectively. The more earlier for injection, the more to prevent the progress of liver fibrosis effectively, the therapeutic effect for advanced liver fibrosis suggests siRNA targeting CTGF treatment of liver fibrosis can become a new target.