人肝内胆管癌相关成纤维细胞的分离、纯化和鉴定及对胆管癌细胞生长和迁移的影响

Isolation,purification and identification of human intrahepatic cholangiocarcinoma-associated fibroblasts and its effect on proliferation and migration of cholangiocarcinoma cells

目的探讨人肝内胆管癌相关成纤维细胞(iCAF)的分离、纯化和鉴定,并初步探究其对肿瘤细胞的影响作用。方法肝内胆管癌标本通过酶消化法(Ⅰ型胶原酶与RPMI 1640混合消化液)分离获得原代细胞,并通过密度梯度离心法进行纯化,iCAF的鉴定通过免疫化学、荧光染色及流式细胞检测进行。通过CCK8及Transwell实验观察iCAF条件培养基对胆管癌细胞的影响作用。结果获得的iCAF以长梭形为主,细胞生长密集相互交织形成网状或漩涡状;免疫细胞化学染色及荧光染色显示:α-SMA阳性、FSP-1阳性;流式细胞检测FSP-1阳性率为98.2%。iCAF条件培养基明显促进了肿瘤细胞增殖和迁移。结论成功分离并纯化了人iCAF,并初步证明iCAF可促进胆管癌细胞增殖和迁移能力,为进一步研究iCAF在肿瘤发生发展中的具体作用机制奠定了重要基础。

Objective To investigate the methods of isolation, purification and identification of human intrahepatic cholangiocarcinoma-associated fibroblasts （iCAFs）, and to explore its impact on cholangiocarcinoma cells. Methods The primary iCAFs were isolated using digestive methods and purified through density gradient centrifugation. Immunocytochemistry, immunofluorescence staining and flow cytometry were performed to confirm iCAFs. Cell counting kit-8 （CCK8） and transwell experiments were performed to assess the effect of iCAFs on cholangiocarcinoma cells. Results The typical morphology of obtained iCAFs were shaped spindle-like, which formed the pattern of net or whirlpool. Typical iCAFs showed positive staining for a-smooth muscle actin and fibroblast-specific protein 1 （FSP-1）,with 98. 2% positive rate of FSP-1. iCAFs conditioned media significantly promoted the proliferation and migration of the cholangiocarcinoma cells. Conclusion Human iCAFs were successfully isolated and identified, which could promote proliferation and migration of the cholangiocarcinoma cells. It provides the basis for further research of iCAFs in the development and progression of intrahepatic cholangiocarcinoma.