化学合成经修饰抗TIMP-2小干扰RNA抑制胆总管结扎大鼠肝纤维化形成

目的研究化学合成经修饰抗金属蛋白酶组织抑制剂-2（tissue式分解inhibitor of metalloproleinase-2，TIMP-2）小干扰RNA（small interfering RNA，siRNA）对胆总管结扎大鼠肝纤维化形成的影响。方法30只雄性SD大鼠随机均分成5组：正常组、假手术组、模型组、阴性对照组、治疗组，行胆总管双重结扎和经肠系膜上静脉（superior mesenter vein，SMV）皮下置管手术，2周后给予治疗组0．05mg·kg^-1 siRNA经皮SMV注射，每3天1次。6周后取所有动物肝组织标本，经门静脉测压（portal vein pressure，PVP）和腹主动脉取血。常规HE染色和Van Gieson（VG）胶原染色，检测血清丙氨酸氨基转移酶（alanine aminotransferase，ALT）、天冬氨酸氨基转移酶（aspartate aminotransferase，AST）和肝组织羟脯氨酸（hydroxyproline，Hyp）含量。应用荧光实时定量PCR法检测TIMP-2、I型胶原纤维（collagen type I，COL I）和Ⅲ型胶原纤维（collagentype Ⅲ，COLⅢ）mRNA的表达。应用Western印迹检测TIMP-2蛋白、α-平滑肌肌动蛋白（α-smooth muscle antibody，α-SMA）和结蛋白（Desmin）的表达。结果经抗TIMP-2 siRNA干预后，大鼠肝脏组织学病变减轻，PVP降低，血清ALT和AST水平下降，肝组织Hyp含量减少，且TIMP-2、COL I和COL Ⅲ mRNA的表达显著低于模型组，TIMP-2蛋白的表达也显著降低。同时肝星状细胞（hepatic stellate cells，HSCs）标记物α-SMA和Desmin蛋白的表达均显著低于模型组，尤以α-SMA蛋白表达减少更为明显。结论化学合成经修饰抗TIMP-2 siRNA能够抑制胆总管结扎大鼠肝纤维化形成，有可能成为肝纤维化治疗的新策略。

Chemically modified siRNA targeting tissue inhibitor of metalloproteinase-2 inhibits hepatic fibro-genesis in bile-duct ligated rats

Objective To study the effects of chemically modified small interfering RNA （siRNA） targeting tissue inhibitor of metalloproteinase-2 （TIMP-2） in rat liver fibrosis by common bile duct ligation. Methods Thirty male rats were randomly divided into five groups： a normal group （n = 6） ,a sham-operated group （n = 6） , a model group （n = 6） , a control siRNA group and a siRNA group. After the 2-weeks common bile duct ligation and percutaneous superior mesenteric vein （SMV） retention catheter, a control siRNA group or a siRNA group was received a dosage of 0.05 mg·kg^-1 control siRNA or siRNA via the SMV every three days for four weeks. All rats were sacrificed after six weeks to measure portal vein pressure （PVP）. The pathological changes of liver tissues were detected by HE and Van Gieson staining. The levels of alanine aminotransferase （ALT） , aspartate aminotransferase （AST） and hydroxyproline （Hyp） were measured, Expression of TIMP-2, collagen type I （COL I） and collagen type Ⅲ （COL Ⅲ） mRNA were evaluated by quantitative real-time PCR. Expression of TIMP-2, α-SMA and Desmin protein were analyzed by Western blotting. Results After treatment of chemically modified siRNA targeting TIMP-2, pathological changes in the siRNA group was markedly attenuated. The levels of PVP, ALT, AST and Hyp in the siRNA group were significantly lower than those in the model group. The mRNA expressions of TIMP-2, COL I and COL III in the siRNA group were decreased significantly higher than that in the model group. Western blotting showed the protein expression of TIMP-2 in the siRNA group was also decreased significantly higher than that in the model group. Furthermore, in the siRNA group, the decrease of α-SMA protein was more obvious than the decrease of Desmin protein although both α-SMA and Desmin protein decreased significantly higher than those in the model group. Conclusion Chemically modified siRNA targeting TIMP-2 inhibits hepatic fibrogenesis in bile-duct ligated rats an