Establishment of a hepatocellular carcinoma cell line with unique metastatic characteristics through in vivo selection and screening for metastasis-related genes through cDNA microarray
Establishment of a hepatocellular carcinoma cell line with unique metastatic characteristics through in vivo selection and screening for metastasis-related genes through cDNA microarray
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DOI：
10.1007/s00432-002-0396-4
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发表时间：
2003-01-01
影响因子：
3.6
通讯作者：
Xue, Q
中科院分区：
文献类型：
--
作者：
Li, Y;Tang, ZY;Xue, Q
目的：目的建立人肝癌肺转移瘤细胞系，为研究肝癌肺转移的分子机制提供合适的模型。研究方法：将肝癌克隆细胞MHCC 97-H接种于BALB/c裸鼠体内，收获肺转移灶，再植入裸鼠体内进行第二轮体内筛选。同样的程序重复两次。建立了来自第三轮肺转移的新细胞系。结果：通过体内筛选，建立了具有独特转移特性的人肝癌细胞系。该细胞系命名为HCCLM 3，为多边形上皮细胞，核型为亚三倍体，群体倍增时间为34.9h。免疫细胞化学法检测细胞甲胎蛋白（AFP）、白蛋白、细胞角蛋白8（CK 8）阳性，B型肝炎表面抗原（HBsAg）阴性。荧光聚合酶链反应（PCR）显示HBV DNA整合在细胞基因组中。当将5x 10（6）个细胞皮下注射到裸鼠中时，致瘤性为100%，潜伏期为11 +/-1天。皮下注射后5周。注射后，肺转移率为100%，肺转移的中位数为每只小鼠121个。裸鼠原位肝移植瘤组织35天后，出现广泛的局部和远处转移，腹壁转移100%，腹腔内转移80%，肝内转移100%，膈转移70%，肺转移100%。肺转移病灶的中位数为每只小鼠268个。利用基因芯片技术比较HCCLM 3与来自同一亲本细胞系但具有低转移潜能的克隆细胞株MHCC 97-L的基因表达谱;共鉴定出25个差异表达基因，其中18个在HCCLM 3中表达降低，7个表达升高，包括细胞周期调控基因Rb 2表达降低，错配修复基因hMSH 2，和信号传导基因蛋白激酶C β 2，并增加信号传导基因MAP激酶、激酶6的表达。结论：一个新的肝癌细胞系，其特征在于通过皮下高肺转移。并建立了原位接种的肝癌转移模型，为肝癌转移的研究提供了新的模型。其基因表达谱有助于了解肿瘤转移的机制，并为抗转移干预提供潜在的靶点。
Purpose: To establish a hepatocellular carcinoma (HCC) cell line from lung metastatic lesions of human HCC in nude mice so as to provide a suitable model for the study of lung-metastasis-related molecular mechanisms. Methods: HCC clone cells MHCC97-H were inoculated into BALB/c nude mice, and the pulmonary metastatic lesions were harvested and re-implanted into nude mice for the second round of in vivo selection. The same procedure was repeated twice. A new cell line from the third round of lung metastases was established. Results: A human HCC cell line with unique metastatic characteristics was established by in vivo selection. This cell line, designated as HCCLM3, was polygonal epithelial cell with hypotriploid karyotype and population doubling time of 34.9 h. The cells were positive for alpha fetoprotein (AFP), albumin, cytokeratin 8 (CK8), and negative for hepatitis B surface antigen (HBsAg) by immunocytochemistry. Fluorescence polymerase chain reaction (PCR) showed HBV DNA integration in the cellular genome. When 5x10(6) cells were injected subcutaneously into nude mice, tumorigenicity was 100%, with a latency period of 11 +/- 1 days. Five weeks after s.c. 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 days, widespread loco-regional and distant metastases occurred, with 100% abdominal wall metastases, 80% intra-abdominal cavity metastases, 100% intrahepatic metastases, 70% diaphragm metastases, and 100% pulmonary metastases. The median number of lung metastatic lesions was 268 per mouse. Gene expression profile of HCCLM3 was compared by cDNA microarray with MHCC97-L, a clonal cell strain from the same parental cell line but with low metastatic potential; 25 differentially expressed genes were identified, 18 of which showed decreased expression and seven increased expression in HCCLM3, including the decreased expression of cell cycle control gene Rb2, mismatch repair gene hMSH2, and signal transduction gene protein kinase C beta2, and increased expression of signal transduction gene MAP kinase, kinase 6. Conclusions: A new HCC cell line characterized by high pulmonary metastases via s.c. and orthotopic inoculation was established, which provides a new model for the study of liver cancer metastasis. Its gene expression profile could help in the understanding of the mechanism of metastasis and provide potential targets for anti-metastasis intervention.