应用SCID鼠筛选肝癌转移性亚克隆及M-H7402亚细胞系的建立

SCREENING OF HUMAN METASTATIC HEPATOMASUB-CLONE BY SCID MICE AND EATABLISHMENT OFTHE M-H7402 SUB-CELL LINE

本研究应用人肝癌细胞SCID鼠转移模型,筛选和建立肝癌转移细胞系,旨在为肝癌转移研究提供实验材料.采用人肝癌细胞H7402,腋后背部皮下接种SCID鼠,接种后66天时,在荷瘤鼠羸弱时引颈处死,取其肺组织,进行原代细胞培养和传代培养,获得肝癌细胞H7402的亚细胞克隆,命名为M-H7402.然后,对M-H7402细胞进行了生物学鉴定,检测了细胞形态、染色体、细胞动力学、细胞周期、甲胎蛋白表达、癌基因表达和转移相关基因表达等指标.结果显示,M-H7402细胞的生长、增殖和形态等与亲本细胞H7402十分相近,其染色体形态仍为人类核型,众数维持在72～80之间,占75.0%.RT-PCR检测结果显示,M-H7402细胞甲胎蛋白表达为阳性.Western blot检测结果显示,与亲本细胞H-7402相比,癌基因C-myc表达水平较高,转移抑制基因nm23的表达水平明显下调.从肺组织中获得的肝癌转移细胞系M-H7402,在体外可连续传代培养,细胞形态不变.应用SCID鼠筛选获得的亚细胞克隆,与亲本细胞形成配对关系,可为肝癌细胞转移的研究提供新的实验材料.

To provide a new material of studying metastasis of hepatocellular carcinoma (HCC) a metastatic human hepatocellular carcinoma cell line was screened and established through severe combined immunodeficiency (SCID) mice. Hepatoma cells, a human H7402 cell line, was inoculated subcutaneously beneath the armpit of SCID mice. The mice were sacrificed after 66 days and the lung tissues of the SCID mice were used to conduct the primary cell culture. A subclone cell line was established and named M-H7402. The M-H7402 cells were characterized in morphology, karyotype, dynamics, flow cytometry, expression of AFP, C-myc and nm23. The results showed that the characteristics of M-H7402 cells were similar with their parental H7402 cells in growth, proliferation and morphology. The M-H7402 cells were hypoteraploid karyotype from human by chromosome analysis, containing 70 - 80 chromosomes in each cell (about 75. 0%. in cell population). AFP mRNA was detectable by RT-PCR in M-H7402 cells. High level of C-myc was showed by Western blot. It was found that nm23 expression was markedly down-regulated in M-H7402 compared with the parental cells. The morphology of M-H7402 cells in culture was maintained, and the cells were available to continually passage. The subclone cell line from its parental cell line, forming a relationship between daughter and parent, provides a new material of studying metastasis of hepatoma.