小鼠酒精性肝纤维化复合模型的建立及肝组织骨桥蛋白和转化生长因子β1的表达  

Establishment of a complex alcoholic liver fibrosis mouse model and investigation of OPN and TGF-pl hepatic expression

作　　者：孔令波[1] 任伟光[1] 米红梅[1] 赵素贤[1] 张玉果[1] 南月敏[1] 

机构地区：[1]河北医科大学第三医院中西医结合肝病科,石家庄050051

出　　处：《中华肝脏病杂志》2013年第3期207-212,共6页Chinese Journal of Hepatology

基　　金：中国肝炎防治基金会王宝恩肝纤维化基金（2009009）

摘　　要：目的快速建立小鼠酒精l生肝纤维化模型，为酒精性肝纤维化发病机制及防治策略的研究奠定基础。方法64只C57BL／6J小鼠随机分为对照组、四氯化碳组、乙醇组、溶剂对照组及乙醇＋四氯化碳组。乙醇＋四氯化碳组小鼠，造模前4周以含40／0乙醇Lieber-DeCarli液体饲料喂养，第5周起联合5％四氯化碳腹腔注射，于造模第0、4、5、6、7、8周各处死6只。其余各组小鼠于造模第8周处死。采用酶法检测小鼠血清ALl7及AST水平lHE及Masson染色观察肝组织病理学变化，并对肝脂肪变、炎症活动及纤维化程度评分。免疫组织化学染色观察肝组织a-平滑肌肌动蛋白（a-SMA）表达变化；实时定量RT-PCR及Westernblot方法检测肝组织骨桥蛋白（OPN）及转化生长因子D1（TGFp1）mRNA及蛋白表达动态变化。结果乙醇＋四氯化碳组小鼠于造模第4周出现轻～中度肝脂肪变，第5周出现炎细胞浸润，窦周出现纤维组织沉积，第6～7周肝小叶内可见点、灶状肝细胞坏死，炎细胞浸润、窦周纤维组织沉积呈进行陛加重，第8周肝细胞片状坏死，桥接纤维化形成。免疫组织化学染色显示：a-SMA主要表达于活化肝星状细胞及纤维组织沉积区域，随造模时间延长表达逐渐增强。乙醇＋四氯化碳组小鼠肝组织OPN表达随造模时间延长逐渐增强，第0、4、5、6、7、8周mRNA相对表达量依次为1．01土0．13、0．80±0．20、1．83土0．25、2．94士0．19、3．45土0．31及5．99士0．17，各组比较，P=476．270，P〈0．01；蛋白相对表达量依次为0．19土0．06、0．48±0．05、0．52士0．06、1．02±0．10、1．52±0．11及1．50±0．08，各组比较，，=298．027，尸〈0．01。TGFp1表达自第5周明显上调，并呈持续高表达，mRNA相对表达量依次为1．03±0．18、1．07±0．23、3．19±0．40、3．31±0．28、1．58±0．18及2．08土0．26，各组比较，F=85．546，P〈0Objective To create a convenient method to establish an alcoholic liver fibrosis model in mice and use it to explore the putative pathogenic mechanisms involving the immunomodulatory proteins osteopontin （OPN） and transforming growth factor-β1 （TGF-β1）. Methods Forty C57BL/6J mice were fed the Lieber-DeCarli 4% ethanol-containing liquid diet for four weeks, followed by an additional four weeks of the 4% ethanol diet combined with intraperitoneal injection of carbon tetrachloride （CC14 5% solution in olive oil; 2ml/ kg body weight, 2 times/week） to induce alcoholic liver fibrosis. Control groups （n = 6 each） included： normal diet; normal diet plus CC14 injections; ethanol diet alone; ethanol diet plus solvent （olive oil） injections. Model establishment was monitored by sacrificing six mice at model inception （week 0）, and weeks 4, 5, 6, 7, and 8 of modeling to collect liver tissues and blood for histological and biochemical analyses. Extent of hepatic steatosis, inflammation, and fibrosis was assessed by hematoxylin-eosin and Masson staining. Liver function markers, serum alanine aminotransferase （ALT） and aspartate aminotransferase （AST） levels, were tested by automated enzymatic assays. Alpha-smooth muscle actin （a-SMA） expression was detected by immunohistochemistry. The mRNA and protein expression of OPN and TGF-f31 was detected by real-time quantitative reverse transcription- PCR and western blotting, respectively. Significance of differences between multiple groups was assessed by one-way ANOVA analysis followed by least significant difference t-test or Kruskal-Wallis H test followed by the Mann-Whitney U test. Results Compared to the control groups, the group of mice administrated ethanol and CC14 developed mild to moderate hepatic steatosis at week 4 of modeling, progressive necroinflammation and perisinusoidal and portal fibrosis from weeks 5-8, and irregular necrosis and bridging fibrosis at week 8. In addition, the model group showed progressive up-regulation of c

关 键 词：肝硬化 骨桥蛋白 转化生长因子p 1 小鼠 模型 动物 酒精 性肝纤维化 

分 类 号：R575.2[医药卫生—消化系统]