抗结缔组织生长因子小分子干扰RNA防治大鼠肝纤维化研究

Small interfering RNA targeting connective tissue growth factor prevents rats liver fibrosis

目的探讨经门静脉注射抗结缔组织生长因子(CTGF)小分子干扰RNA(siRNA)是否能在体内抑制大鼠肝CTGF基因表达及防治大鼠肝纤维化。方法雄性SD大鼠24只,均分成4组,模型组皮下注射40%CCl4(3ml/kg)及门静脉注射生理盐水,每3d1次,连续6周;预防组皮下注射CCl4及门静脉注射抗CTGFsiRNA(0.1mg/kg),每3d1次,连续6周;治疗组皮下注射CCl42周,随后再给予抗CTGFsiRNA及CCl44周;对照组门静脉注射生理盐水6周。于最后1次CCl4注射后3d行门静脉测压,并取血及组织标本,检测血清转氨酶、透明质酸(HA)及Ⅲ型前胶原(PⅢNP)浓度,应用RTPCR及Western印迹法检测CTGFmRNA及蛋白质在大鼠肝组织表达,应用HE及Siriusred胶原染色检测肝组织炎症及纤维化,应用偏光扫描对肝组织胶原染色面积进行定量。结果与模型组相比,预防组及治疗组大鼠肝组织CTGFmRNA及蛋白质表达显著下调,肝组织炎症、坏死及纤维化明显减轻,血清转氨酶、HA及PⅢNP浓度显著降低;预防组、模型组和治疗组的肝组织胶原染色面积分别为5.8%±0.8%、12.3%±0.8%和7.2%±0.9%(P<0.01);门静脉压力分别为(14.2±2.3)cmH2O、(20.6±5.8)cmH2O和(15.1±3.6)cmH2O(P<0.05),明显降低。结论经门静脉注射抗CTGFsiRNA能在体内显著抑制大鼠肝CTGF基因表达并能有效防治大鼠肝纤维化,提示抗CTGFsiRNA有潜力成为防治肝纤维化的一种新策略。

Objective To explore whether portal vein 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 Twenty four male rats were equaly divided into four groups. Rats received subcutaneous injection of 40% CCl4 (3 ml/kg) together with portal vein injection of saline every three days for 6 consecutive weeks were served as model group; CCl4 together with portal vein delivery of siRNA (0.1 mg/kg) as preventive group; CCl4 for 2 weeks followed by CCl4 and CTGF siRNA for more than 4 weeks as curative group, and only intraportal injection of saline as control group. Portal vein pressure in all rats at 3 days after the last CCl4 injection were measured, and blood and hepatic tissue from rats were harvested. Serum concentration of transaminases, hyaluronic acid(HA) and procollagen type Ⅲ (PⅢNP) were measured. Expression of CTGF mRNA and protein in rats liver was evaluated by RT-PCR and Western blot, respectively. Inflammation and fibrosis in rats liver was analyzed by H-E and Sirius red collagen staining. The fibrotic area was measured quantitatively by a computer-aided manipulator. Results Compared with model group, the expression of CTGF mRNA and protein in liver in both preventive and curative groups were markedly down-regulated. Inflammation, necrosis and fibrosis in hepatic tissue was significantly attenuated. In addition, the serum concentration of transaminases, HA and PⅢNP were greatly reduced. The fibrotic areas in preventive, curative and model groups were 5.8%±0.8%, 7.2%±0.9% and 12.3%±0.8%,respectively (P<0.01), while the portal vein pressure were (14.2 ± 2.3 ), (15.1 ± 3.6 ) and ( 20.6 ± 5.8) cm H2O ,respectively (P<0.01). Conclusions Portal vein delivery of CTGF siRNA significantly inhibit CTGF expression in rats liver in vivo, and effectively prevent rats hepatic fibrosis, suggesting siRNA targeting CTGF has the potential to be a novel strategy for anti-liver fibrosis.