[通过体内筛选建立具有自发肺转移的人肝癌细胞系]

[Establishment of human hepatocellular carcinoma cell line with spontaneous pulmonary metastasis through in vivo selection].

作者信息

Li Yan, Tang Zhaoyou, Ye Shenglong, Liu Yinkun, Chen Jie, Xue Qiong, Huang Xiaowu, Chen Jun, Bao Weihua, Yang Jiong, Gao Dongmei

机构信息

Liver Cancer Institute and Zhong Shan Hospital of Fudan University, Shanghai, 200032, China.

出版信息

Zhonghua Yi Xue Za Zhi. 2002 May 10;82(9):601-5.

PMID:12133480

Abstract

OBJECTIVE

To establish a hepatocellular carcinoma (HCC) cell line from lung metastatic lesions of human HCC in nude mice so as to provide suitable model for the study of lung-metastasis-related molecular mechanisms.

METHODS

HCC clone cells MHCC97-H were inoculated subcutaneously into nude mice. The pulmonary metastatic lesions were harvested from the moribund animals and then re-implanted into the nude mice for the second round of selection. The same procedure was repeated twice. New cell line from the third round of lung metastasis was thus established and the following parameters were studied: morphology, in vitro growth curve, plate efficiency, migration, karyotype, flow cytometry, immunocytochemistry for AFP, albumin and cytokeratin 8 (CK8), flourescent PCR for HBV DNA, tumorigenicity and spontaneous metastasis via subcutaneous injection and orthotopic implantation of tumor tissue.

RESULTS

The cell line established from nude mouse lung metastasis was designated as HCCLM3, consisting of polygonal epithelial cells with hypotriploid karyotype, its main range of chromosome being 55 - 58. The cell population doubling time was 34.9 hours, plate efficiency 32.4 +/- 3.2%, and cell migration rate 20 +/- 2 microm/h. The cells were positive for AFP, albumin, CK8, and P16 and negative for P53, nm23 and HBsAg. Fluorescent PCR showed HBV DNA integration into cellular genome. When 5 x 10(6) cells were injected subcutaneously into nude mice, the tumorigenicity was 100% with a latency period of 11 +/- 1 d. Five weeks after subcutaneous injection, the pulmonary metastatic rate was 100%, the median number of lung metastases being 121 per mouse. After orthotopic implantation of tumor tissue into nude mouse liver for 35 d, wide spread regional and distant metastases occurred, the metastatic rate was 100% for abdominal wall, 80% for organs in abdominal cavity, 100% for liver, 70% for diaphragm, and 100% for lung. The median number of lung metastatic lesions was 268 per mouse.

CONCLUSION

A new HCC cell line has been established, which is characterized by high pulmonary metastasis via both subcutaneous and orthotopic inoculation. It provides a new model for the study of liver cancer metastasis.

摘要

目的

从人肝癌裸鼠肺转移瘤中建立肝癌细胞系，为肝癌肺转移相关分子机制的研究提供合适模型。

方法

将肝癌克隆细胞MHCC97-H皮下接种于裸鼠。从濒死动物获取肺转移瘤，再接种于裸鼠进行第二轮筛选。重复上述过程两次。由此建立第三轮肺转移的新细胞系，并研究其形态学、体外生长曲线、平板接种效率、迁移能力、核型、流式细胞术、甲胎蛋白（AFP）、白蛋白及细胞角蛋白8（CK8）免疫细胞化学、乙肝病毒（HBV）DNA荧光定量聚合酶链反应（PCR）、致瘤性以及通过肿瘤组织皮下注射和原位植入的自发转移情况。

结果

从裸鼠肺转移建立的细胞系命名为HCCLM3，由多角形上皮细胞组成，核型为亚三倍体，其染色体数主要范围为55 - 58。细胞群体倍增时间为34.9小时，平板接种效率为32.4±3.2%，细胞迁移率为20±2微米/小时。细胞AFP、白蛋白、CK8及P16呈阳性，P53、nm23及乙肝表面抗原（HBsAg）呈阴性。荧光定量PCR显示HBV DNA整合入细胞基因组。当将5×10⁶个细胞皮下注射入裸鼠时，致瘤率为100%，潜伏期为11±1天。皮下注射5周后，肺转移率为100%，每只小鼠肺转移灶中位数为121个。将肿瘤组织原位植入裸鼠肝脏35天后，出现广泛的局部和远处转移，腹壁转移率为100%，腹腔脏器转移率为80%，肝脏转移率为100%，膈肌转移率为70%，肺转移率为100%。每只小鼠肺转移灶中位数为268个。