Hedgehog通路介导上皮-间质转化促进CCl_(4)诱导的大鼠肝纤维化进展  

Hedgehog Signaling Pathway Mediates Epithelial-mesenchymal Transition and Promotes CCl_(4)-induced Liver Fibrosis in Rats

作　　者：张婷婷 严峻彬 叶蕾[1] 毛晓娟[1] 严茂祥[1] 陈芝芸[1] ZHANG Tingting;YAN Junbin;YE Lei(The First Affiliated Hospital of Zhejiang Chinese Medical University,Hangzhou(310006),China)

机构地区：[1]浙江中医药大学附属第一医院,杭州310006

出　　处：《浙江中医药大学学报》2021年第4期345-352,共8页Journal of Zhejiang Chinese Medical University

基　　金：浙江省自然科学基金项目(LY14H270010)。

摘　　要：[目的]观察四氯化碳(carbon tetrachloride,CCl4)诱导大鼠肝纤维化发生发展过程中,Hedgehog信号通路及上皮-间质转化(epithelial-mesenchymal transition,EMT)关键分子的变化情况,探讨其可能的作用机制。[方法]将64只雄性无特殊病原体(specific pathogen free,SPF)级SD大鼠随机分为正常组和模型组,每组32只。模型组大鼠皮下注射橄榄油稀释的40%CCl43mL·kg-1,正常组用等容量的橄榄油代替CCl4,2次/周,首次加倍,共6周,建立大鼠肝纤维化模型,于2、7、21、42d采用全自动生化仪检测血清谷丙转氨酶(alanine aminotransferase,ALT)、谷草转氨酶(aspartate aminotransferase,AST)、碱性磷酸酶(alkaline phosphatase,ALP)、总胆红素(total bilirubin,TBIL)水平,以碱水法检测肝组织羟脯氨酸(hydroxyproline,Hyp)含量,苏木精-伊红(hematoxylin-eosin,HE)染色和Masson染色观察肝组织炎症及纤维化程度,实时荧光定量聚合酶链式反应(quantitative Real-time polymerase chain reaction,qRT-PCR)和Western blot检测肝组织Hedgehog信号通路及EMT关键分子上皮-钙粘素(epithelial-cadherin,E-cadherin)、α-平滑肌肌动蛋白(α-smooth muscle actin,α-SMA)、平滑受体(smoothened,Smo)及神经胶质瘤相关癌基因同源蛋白-1(glioma-associated oncogene homolog-1,Gli-1)mRNA和蛋白表达。[结果]模型组大鼠血清ALT、AST、ALP、TBIL水平和肝组织Hyp含量随时间延长逐渐增高,21、42d均显著高于正常组同期(P<0.01)。肝组织炎症和纤维化程度也随时间延长而逐渐加重,均较正常组同期显著增高(P<0.05,P<0.01)。模型组肝脏E-cadherin mRNA和蛋白表达随时间延长而逐渐下降,α-SMA、Smo、Gli-1的mRNA和蛋白表达则逐渐上升(P<0.05,P<0.01),且21、42d与正常组同期差异均有统计学意义(P<0.05,P<0.01)。[结论]CCl4诱导的大鼠肝纤维化发生过程中存在EMT和Hedgehog信号通路异常激活,Hedgehog信号通路可能通过介导EMT,从而促进肝纤维化进展。[Objective]To observe the changes of Hedgehog signaling pathway and key molecules of epithelial-mesenchymal transition(EMT)during the development of carbon tetrachloride(CCl4)-induced liver fibrosis in rats,and to explore its possible mechanism.[Methods]Sixty-four male specific pathogen free(SPF)SD rats were randomly divided into normal group and model group,with 32 rats in each group.The rat liver fibrosis model was induced by subcutaneous injection of 40%CCl4 olive oil dilution at 3mL·kg-1;in normal group 40%CCl4 was replaced by equal volume of olive oil twice a week,the first time doubled,for 6 consecutive weeks.At the 2nd,7th,21st and 42nd day,the serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST),alkaline phosphatase(ALP),total bilirubin(TBIL)were detected by fully automatic biochemical analyzer,the hydroxyproline(Hyp)content of liver tissues were tested with Caustic method,the inflammation and fibrosis in liver tissue were observed with hematoxylin-eosin(HE)and Masson staining,the mRNA and protein expression of the key molecules of the Hedgehog signaling pathway and EMT in liver tissue,epithelial cadherin(E-cadherin),α-smooth muscle actin(α-SMA),smoothened(Smo),glioma-associated oncogene homolog-1(Gli-1)were detected with quantitative Real-time polymerase chain reaction(qRT-PCR)and Western blot.[Results]The serum ALT,AST,ALP,TBIL levels and the Hyp content of liver tissue in model group increased gradually as time went on,and the two time points of 21st and 42nd day were significantly higher than normal group at the same time point(P<0.01).The inflammation and fibrosis of liver tissue also increased gradually as time went on,were higher than normal group at the same time(P<0.05,P<0.01).The E-cadherin mRNA and protein expression in liver tissue decreased gradually as time went on,while the mRNA and protein expression ofα-SMA,Smo and Gli-1 increased gradually(P<0.05,P<0.01),and the phase points at 21st and 42nd day were statistically different from those of normal group at the s

关 键 词：肝纤维化 HEDGEHOG信号通路 上皮-间质转化 CCl_(4) 大鼠 炎症 上皮细胞 间质细胞 

分 类 号：R331[医药卫生—人体生理学]