目的观察胆管结扎诱导肝纤维化大鼠肝细胞是否发生上皮间质表型变化。方法 24只Wistar大鼠分为假手术组和胆管结扎组,运用HE及Massion染色检测肝纤维化变化;免疫印迹方法检测大鼠肝脏中上皮标志E-钙蛋白、白蛋白和I型胶原、波形蛋白的表达;采用免疫荧光双染的方法检测成纤维细胞特异性蛋白(FSP-1)+波形蛋白;FSP-1+I型胶原;FSP-1+白蛋白;白蛋白+I型胶原等蛋白在细胞中的定位。结果胆管结扎组大鼠的ISHAK纤维化评分(4.42±1.16 vs 0.00±0.00,P=-0.000),METAVIR纤维化评分(3.42±0.67 vs 0.00±0.00,P=-0.000)及Massion染色中胶原含量(0.30±0.06 vs 0.11±0.07,P=-0.000)较假手术组明显增高。与假手术组相比,胆管结扎组中上皮标志白蛋白(0.53±0.63 vs 1.12±0.01,P=0.000)、E-钙蛋白(0.21±0.01 vs 0.44±0.01,P=0.000)明显减少,而I型胶原(8.21±0.12 vs 0.24±0.01,P=-0.000)、波形蛋白(3.14±0.01 vs 0.37±0.01,P=0.000)显著增高。胆管结扎组中共表达FSP-1+波形蛋白、FSP-1+I型胶原、FSP-1+白蛋白、白蛋白+I型胶原的细胞较假手术组明显增多。结论在胆管结扎组诱导的肝纤维化大鼠肝脏中上皮标记减少,间质标志增加,提示有部分上皮细胞可能发生了上皮间质表型转化。
Objective To investigate the occurrence of epithelial to mesenchymal phenotype transition(EMT) in the liver of rats following bile duct ligation(BDL).Methods Twenty-four male Wistar rats were randomized into sham-operated group and BDL group.Liver fibrosis of the rats was evaluated by HE staining and Masson's trichrome staining.Western blotting was used to detect the expression levels of the epithelial markers albumin and E-cadherin and the mesenchymal markers typeⅠcollagen and vimentin in the liver tissue.Immunofluorescence was employed to determine the co-localizations of FSP-1+vimentin, FSP-1+typeⅠcollagen,FSP-1+albumin,and albumin+typeⅠcollagen in cells.Results Compared with those in sham-operated group,the rats in BDL group showed significantly increased ISHAK fibrosis score(4.42±1.16 vs 0,P=0.000),METAVIR fibrosis score(3.42±0.67 vs 0,P=0.000) and typeⅠcollagen levels(0.30±0.06 vs 0.11±0.07,P=0.000) with up-regulated protein levels of albumin(0.53±0.63 vs 1.12±0.01,P=0.000) and E-cadherin(0.21±0.01 vs 0.44±0.01,P=0.000) and down-regulated typeⅠcollagen (8.21±0.12 vs 0.24±0.01,P=0.000) and vimentin(3.14±0.01 vs 0.37±0.01,P=0.000).The number of cells with co-localizations of FSP-1+vimentin,FSP-1+typeⅠcollagen,FSP-1+albumin,and albumin+typeⅠcollagen was also significantly increased in BDL group.Conclusion BDL causes significantly decreased expression of epithelial markers and increased expressions of the mesenchymal markers in rats,indicating the occurrence of EMT in some of the liver cells.