摘要
目的建立二乙基亚硝胺(diethylinitrosamine,DEN)诱导大鼠肝纤维化-肝癌模型,检测肝组织中磷酸化Smad3连接区(linker-phosphorylated Smad3)和纤溶酶原激活物抑制剂-1(plasminogen activator inhibitor 1,PAI-1)蛋白的表达。方法♂SD大鼠50只随机分为DEN组和正常对照组,DEN组给予0.2%DEN溶液灌胃,每周5次至14周。对照组给予溶媒。分别于4、8、12、16周结束时处死动物。进行肝组织苏木精-伊红(HE)染色和Van Gieson(VG)胶原纤维染色;分别采用免疫组化法和Western blot法检测肝组织中pSmad3L及PAI-1蛋白的表达。结果对照组肝组织结构正常,DEN组4周时,肝细胞点状坏死及炎性细胞浸润;8周时,肝细胞严重脂肪变性、灶性坏死,门管区出现纤维组织增生;12周时,增生的胶原束包绕并分割肝小叶,正常的肝小叶结构被破坏,假小叶形成;到16周结束时,剩余存活大鼠均形成肝癌,肝癌细胞排列成条索状或团块状,异型性明显。免疫组化结果显示,pSmad3L和PAI-1在正常肝组织中几乎无表达,DEN组4周时肝组织中有少量阳性表达,8周和12周表达量逐渐增强,到16周时达到高峰,尤其是在肿瘤边界区域肝组织的pSmad3L和PAI-1表达更强。Westernblot结果显示,pSmad3L和PAI-1表达也逐渐增强,与免疫组化结果基本一致。结论 DEN诱导大鼠肝纤维化-肝癌动态过程中,pSmad3L和PAI-1蛋白的表达逐渐增强,提示其损伤机制可能涉及TGF-β/Smads/PAI-1通路。
Aim To establish diethylinitrosamine（DEN）-induced hepatic fibrosis-carcinoma model in rat and measure linker-phosphorylated Smad3（pSmad3L） and plasminogen activator inhibitor-1（PAI-1） protein in liver tissues.Methods Fifty male Sprague-Dawley rats were randomly divided into DEN group and normal control group.The rats of DEN group were administrated 0.2% DEN solution by gavage five times a week for 14 weeks,and the control group received vehicle.The animals were sacrificed at the end of 4th,8th,12th,16th week,respectively.Liver tissues were stained with hematoxylin-eosin（HE） and Van Gibson ’s collagen fiber stain（V.G） for histopathologic examination.The protein expression of pSmad3L and PAI-1 was measured by immunohistochemical method and Western blot method.Results In DEN group,spotty necrosis of hepatocytes and inflammatory cell infiltration occurred in hepatic lobule on 4th week.Severe hepatic steatosis and focal necrosis of hepatocytes were observed on 8th week,accompanied with collagen fiber hyperplasia in portal area.On 12th week,hepatic lobules were encysted and separated by hyperplastic collagen bundles.The normal structure of lobules was destroyed,and pseudo lobules formed.At the end of 16th week,the remaining surviving rats all developed hepatocarcinoma.The hepatoma cells were arranged in cords or mass with obvious atypia.The structure of liver tissue was normal in control group.There was almost no positive expression of pSmad3L and PAI-1 in normal liver tissues.In DEN group,the liver tissues were observed with weak positive expression of pSmad3L and PAI-1 on 4th week.The amount and intensity of positive expression gradually increased from 8th to 12th week and reached maximum to 16th week.In addition,pSmad3L and PAI-1 were both highly expressed in the region of tumor boundary.Furthermore,the expression of pSmad3L and PAI-1 protein,which was similar to the results by immunohistochemical methods,enhanced gradually from 4th to 16th week in rats induced by DEN.Conclusion During the process of DEN induced hepatic fibrosis-carcinoma in rats,the expression of pSmad3L and PAI-1 gradually increases,suggesting the injury mechanisms of DEN may involve TGF-β / Smads / PAI-1 pathway.
出处
《中国药理学通报》
CAS
CSCD
北大核心
2012年第10期1393-1397,共5页
Chinese Pharmacological Bulletin
基金
国家自然科学基金资助项目(No 81073098)
