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

肝脏 ›› 2017, Vol. 22 ›› Issue (6): 517-522.

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

沙朦,Jeong Seogsong,顾劲扬,张建军,夏强

200127 上海交通大学医学院附属仁济医院肝脏外科

出版日期:2017-06-30 发布日期:2017-06-30
通讯作者: 夏强,Email:xiaqiang@medmail.com.cn
基金资助:
国家自然科学基金(81472243)

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

SHA Meng, JEONG Seog-song, GU Jin-yang, ZHANG Jian-jun, XIA Qiang

Department of Liver Surgery, Renji Hospital, School of Medicine, Shanghai Jiaotong University, Shanghai 200127, China

Online:2017-06-30 Published:2017-06-30
Contact: XIA Qiang, Email: xiaqiang@medmail.com.cn

摘要/Abstract

摘要： 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。

Abstract: 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 α-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.

Key words: Intrahepatic cholangiocarcinoma, Cancer-associated fibroblasts, Isolation, Purification, Identification, Proliferation, Migration

沙朦,Jeong Seogsong,顾劲扬,张建军,夏强. 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[J]. 肝脏, 2017, 22(6): 517-522.

SHA Meng, JEONG Seog-song, GU Jin-yang, ZHANG Jian-jun, XIA Qiang. Isolation, purification and identification of human intrahepatic cholangiocarcinoma-associated fibroblasts and its effect on proliferation and migration of cholangiocarcinoma cells[J]. Chinese Hepatolgy, 2017, 22(6): 517-522.

参考文献 18

[1]	Moeini A,Sia D,Bardeesy N,et al. Molecular pathogenesis and targeted therapies for intrahepatic cholangiocarcinoma. Clin Cancer Res,2016,22:291-300.
[2]	Hui L,Chen Y.Tumor microenvironment: Sanctuary of the devil. Cancer Lett,2015,368:7-13.
[3]	Nakamura H,Arai Y,Shibata T,et al.Genomic spectra of biliary tract cancer.Nat Genet,2015,47: 1003-1010.
[4]	Lee JI,Campbell JS.Role of desmoplasia in cholangiocarcinoma and hepatocellular carcinoma. J Hepatol,2014,61:432-434.
[5]	Shiga K,Hara M,Nagasaki T,et al.Cancer-associated fibroblasts: their characteristics and their roles in tumor growth. Cancers (Basel),2015,7:2443-2458.
[6]	Bauer M,Su G,Casper C,et al.Heterogeneity of gene expression in stromal fibroblasts of human breast carcinomas and normal breast.Oncogene,2010,29:1732-1740.
[7]	Ivey JW,Bonakdar M,Kanitkar A,et al.Improving cancer therapies by targeting the physical and chemical hallmarks of the tumor microenvironment.Cancer Lett,2016,380:330-339.
[8]	司徒镇强,吴君正.细胞培养. 西安:世界图书出版公司,2007.
[9]	杨晓柳,高强.肝内胆管癌相关成纤维细胞的分离、纯化及鉴定.中国临床医学,2016, 23:2.
[10]	Sharon Y,Alon L,Glanz S,et al.Isolation of normal and cancer-associated fibroblasts from fresh tissues by Fluorescence Activated Cell Sorting (FACS).J Vis Exp,2013, 71:e4425.
[11]	Luo HJ,Tu G,Liu ZM,et al.Cancer-associated fibroblasts::A multifaceted driver of breast cancer progression.Cancer Lett,2015,361:155-163.
[12]	Yu T,Guo Z,Wang Q,et al.Cancer-associated fibroblasts promote non-small cell lung cancer cell invasion by upregulation of glucose-regulated protein 78 (GRP78) expression in an integrated bionic microfluidic device.Oncotarget,2016,7:25593-25603.
[13]	Goicoechea SM,Garcia-Mata R,Staub J,et al.Palladin promotes invasion of pancreatic cancer cells by enhancing invadopodia formation in cancer-associated fibroblasts.Oncogene,2014,33:1265-1273.
[14]	Sun C,Fukui H,Miwa H,et al.FGF9 from cancer-associated fibroblasts is a possible mediator of invasion and anti-apoptosis of gastric cancer cells.BMC Cancer,2015,15:333.
[15]	Hawinkels LJ,Paauwe M,Sier CF,et al.Interaction with colon cancer cells hyperactivates TGF-β signaling in cancer-associated fibroblasts.Oncogene,2014,33:97-107.
[16]	Yu Y,Xiao CH,Tan LD,et al.Cancer-associated fibroblasts induce epithelial-mesenchymal transition of breast cancer cells through paracrine TGF-beta signaling.Br J Cancer,2014,110: 724-732.
[17]	Yamamura Y,Asai N,Enomoto A,et al.Akt-Girdin signaling in Cancer-Associated fibroblasts contributes to tumor progression.Cancer Res,2015,75:813-823.
[18]	Li F,Zhu YT.HGF-activated colonic fibroblasts mediates carcinogenesis of colonic epithelial cancer cells via PKC-cMETERK1/2-COX-2 signaling.Cell Signal,2015,27:860-866.