人增生肝外胆管上皮细胞的原代培养及组织学特点

目的 建立人增生胆管上皮细胞(BECs)的原代培养.方法 通过胶原酶消化、机械分离对不同原因引起的扩张人肝外胆管上皮细胞进行分离纯化,建立原代培养;利用免疫细胞化学和免疫荧光染色,对培养的胆管上皮细胞表达CK-19、E-cadherin、Vimentin、α-SMA和S100A4进行特性鉴定.结果 BECs的贴壁成活率高,原代培养细胞生长速度快;免疫细胞化学和免疫荧光染色显示,上皮细胞特异性标志CK-19和E-cadherin表达阳性,间质细胞标志Vimentin表达弱阳性或阴性,α-SMA表达阴性,但上皮间质表型转变标志蛋白S100A4呈阳性表达.结论 本方法经济、简便、易行,在体外成功分离培养增生的人肝外胆管上皮细胞,获得了高纯度的BECs(>98%);发现增生的胆管上皮细胞发生了上皮间质表型转变,可能是参与胆汁性肝纤维化的机制之一.

Primary culture and characterization of human hyperplastic extrahepatic biliary epithelial cells

Objective Establishing the primary culture of human hyperpiastic biliary epithelial cells (BECs). Methods Cells were isolated by digestion with collagenase followed by scraping of the mu-cosa of human dilated extrahepatic bile duct. The characteristic and purity of the epithelial cells in primary culture were confirmed by immunocytochemistry and immunofluorescence with antibodies against cytokeratin 19 (CK-19) ,E-cadherin,Vimentin,α-SMA and S100A4. Results The BECs primary culture expanded quickly after attachment;Greater than 95% of the cells were positive for biliary epithelium marker CK-19 and E-cadherin,but weak positive or negative for vimentin,a marker for mesenchymal cells;The BECs were also negative for α-SMA,but positive for the epithelial-to-mesenchymal transition (EMT) marker S100A4. Conclusion We present an economic and easy-performing method for the isolation and culture of human extrahepatic BECs. These results indicate that highly purified human hyperplasic extrahepatic BECs can be prepared successfully(>98% ). And these cells were found to undergo EMT. It is suggested that the EMT of BECs contributes to liver biliary fibrogenesis.