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中华肝脏外科手术学电子杂志

中华肝脏外科手术学电子杂志 ›› 2019, Vol. 08 ›› Issue (03) : 265 -269. doi: 10.3877/cma.j.issn.2095-3232.2019.03.020

所属专题： 文献；

基础研究

肝癌实质细胞及肿瘤相关成纤维细胞分离培养方案的优化
向俊西1, 刘鹏1, 田波彦2, 李青山1, 杨丽斐3, 郑幸龙3, 张谞丰1, 刘学民1, 吕毅1,†()   
  1. 1. 710061　西安交通大学第一附属医院精准外科与再生医学国家地方联合工程研究中心；710061　西安交通大学第一附属医院肝胆外科
    2. 710061　西安交通大学第一附属医院肝胆外科
    3. 710061　西安交通大学第一附属医院精准外科与再生医学国家地方联合工程研究中心
  • 收稿日期:2019-02-25 出版日期:2019-06-10
  • 通信作者: 吕毅
  • 基金资助:
    国家自然科学基金(81501608)

Optimization of isolation and culture of primary liver cancer parenchyma cells and cancer-associated fibroblasts

Junxi Xiang1, Peng Liu1, Boyan Tian2, Qingshan Li1, Lifei Yang3, Xinglong Zheng3, Xufeng Zhang1, Xuemin Liu1, Yi Lyu1,†()   

  1. 1. National-Local Joint Engineering Research Center for Precise Surgery and Regenerative Medicine, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China; Department of Hepatobiliary Surgery, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China
    3. National-Local Joint Engineering Research Center for Precise Surgery and Regenerative Medicine, the First Affiliated Hospital of Xi'an Jiaotong University, Xi'an 710061, China
  • Received:2019-02-25 Published:2019-06-10
  • Corresponding author: Yi Lyu
  • About author:
    Corresponding author: Lyu Yi, Email:
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引用本文：

向俊西, 刘鹏, 田波彦, 李青山, 杨丽斐, 郑幸龙, 张谞丰, 刘学民, 吕毅. 肝癌实质细胞及肿瘤相关成纤维细胞分离培养方案的优化[J]. 中华肝脏外科手术学电子杂志, 2019, 08(03): 265-269.

Junxi Xiang, Peng Liu, Boyan Tian, Qingshan Li, Lifei Yang, Xinglong Zheng, Xufeng Zhang, Xuemin Liu, Yi Lyu. Optimization of isolation and culture of primary liver cancer parenchyma cells and cancer-associated fibroblasts[J]. Chinese Journal of Hepatic Surgery(Electronic Edition), 2019, 08(03): 265-269.

目的

分离培养人原发性肝癌（肝癌）实质细胞及肿瘤相关成纤维细胞（CAF），并检测其生物学特性。

方法

标本来源于2015年9月至2016年10月西安交通大学第一附属医院15例肝癌患者手术切除新鲜肝癌组织。其中男12例，女3例；年龄28～64岁，中位年龄45岁；肝细胞癌13例，胆管细胞型肝癌2例。患者均签署知情同意书，符合医学伦理学规定。采用胶原酶消化法分离培养人原代肝癌细胞及CAF。倒置显微镜下观察肝癌实质及CAF的形态特征。CCK-8法测定细胞活性，免疫荧光鉴定肝癌细胞标志物AFP及CAF标志物波形蛋白（Vimentin）表达。

结果

原代肝癌实质细胞培养成功率为2/15，CAF培养成功率为11/15，均为肝细胞癌。肝癌实质细胞为上皮样细胞，铺路石样分布。CAF为长梭形或多角形，呈"鱼群样"分布。肝癌细胞与CAF生长状态良好，增殖活跃，呈对数型生长，CAF的增殖能力略强于肝癌细胞。免疫荧光染色显示原代肝癌细胞质中AFP染色阳性，CAF中Vimentin蛋白表达阳性。

结论

采用胶原酶消化法可以成功获得高增殖活性的人原代肝癌实质细胞及CAF。

关键词: 癌，肝细胞, 成纤维细胞, 培养技术
Objective

To isolate and culture human primary liver cancer (PLC) parenchyma cells and cancer-associated fibroblasts (CAF) and to investigate the biological characteristics.

Methods

Fresh PLC tissues were collected from 15 patients with PLC who underwent surgical resection in the First Affiliated Hospital of Xi'an Jiaotong University from September 2015 to October 2016. Among them, 12 cases were male and 3 female, aged 28-64 years with a median of 45 years old. 13 cases were diagnosed with hepatocellular carcinoma and 2 cases of cholangiocarcinoma. The informed consents of all patients were obtained and the local ethical committee approval was received. Primary human hepatoma cells and CAF were isolated and cultured with collagenase digestion. The morphological characteristics of PLC parenchyma cells and CAF were observed under inverted microscope. The cell viability was assessed by CCK-8 assay. The expression levels of PLC cell marker alpha-fetoprotein (AFP) and CAF marker Vimentin were measured by immunofluorescence staining.

Results

The success rate of primary PLC parenchymal cells culture was 2/15, and 11/15 for CAF culture, all of which were hepatocellular carcinoma. The PLC parenchymal cells were epithelioid cells, which were distributed in a paving stone pattern. CAF was in long fusiform or polygonal shape, distributed in a fish swarm manner. PLC cells and CAF grew and proliferated well in the logarithmic growth phase. The proliferation ability of CAF was slightly stronger than that of PLC cells. Positive AFP was observed in the cytoplasm of primary PLC cells by immunofluorescence staining and positive Vimentin protein expression was observed in CAF.

Conclusions

Human primary PLC parenchyma cells and CAF with high proliferation can be successfully obtained through collagenase digestion.

Key words: Carcinoma, hepatocellular, Fibroblasts, Culture techniques
图1 原代肝癌细胞及CAF形态
图3 原代肝癌细胞及CAF免疫荧光染色
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