梯度密度离心法分离肝癌细胞及其转移性细胞株建立.docVIP

梯度密度离心法分离肝癌细胞及其转移性细胞株建立 　 作者：张萌， 胡少为， 姜桔红， 文剑明 【摘要】 【目的】 建立新的肝细胞癌细胞系，为肝癌实验研究提供更多纯化癌细胞。【方法】 从肝细胞癌患者原发灶和门脉癌栓中获取癌组织，采用Percoll浓度梯度分离癌细胞进行培养，用微环境消化法获取癌细胞克隆并建立细胞系，对培养成功的细胞系进行生长曲线测定、染色体G带核型分析、对比基因组杂交和裸鼠接种成瘤实验。【结果】 对20例肝癌组织进行癌细胞分离培养和克隆，成功建立2例细胞系H2M和H4M，这2株细胞均来自门脉癌栓，它们的染色体数目测定均为超3倍体（71～78条）。CGH检测显示H2M主要的细胞遗传学改变为染色体4q、13q、16q、17p和19p缺失和1q、3q、5p、6p、7q和8q扩增，其中有一条标记染色体含有1q和6p［t(1;6)染色体］；而H4M染色体8p, 9, 13q, 16q 缺失和6p, 7p, 11p, 11q13扩增，其中有一条标记染色体含有一长的均染区（hsr）。H2M细胞接种裸鼠1个月，产生典型的人肝细胞癌，但H4M接种尚未能成瘤。【结论】Percoll浓度梯度分离癌细胞和微环境消化法取得癌细胞克隆是建立原代细胞系的有效方法。2例肝癌细胞系的建立为以后肝癌研究提供了实验材料，所测出的肝癌细胞系的细胞遗传学改变及在裸鼠接种成瘤，为肝癌细胞癌基因和抑癌基因筛选提供了研究线索和实验模型。 【关键词】 肝癌； 细胞株； 核型； 比较基因组杂交 Abstract:【Objective】To establish hepatocellular carcinoma (HCC) cell lines and provide more pure liver carcinoma cells for HCC studies.【Methods】The carcinoma tissues were obtained from the primary lesion and metastatic lesion in portal vein of HCC. The carcinoma cells were isolated by Percoll density gradient centrifuge and cultivated. The cell clones were then obtained by micro-environmental digestion and cell lines were established. The growth curves, G band chromosome karyotype, comparative genomic hybridization (CGH) of the established cells were characterized. The cells were implanted in nude mice for tumorigenesis.【Results】 20 cases of HCC cells were isolated and cultivated. Two cell lines (designated as H2M and H4M) were successfully established from 2 cases of emboli of portal vein. The karyotype showed that both cell lines are a hypertriploid (71～ 78 chromosomes). A marker chromosome containing 1q and 6p ［t(1;6)］ was found in H2M cells and a huge marker chromosome containing a long homogeneously staining region (hsr) in H4M cells. The main genetic alterations analyzed by CGH were a high copy number amplification of 1q, 3q, 5p, 6p, 7q and 8q and loss of 4q, 13q, 16q, 17p 19p in H2M cells, whereas, amplification of 6p, 7p, 11p, 11q13 and loss of 8p, 9, 13q, 16q in H4M cells. Implantation of H2M cells in nude mice for a month produ