大鼠颊黏膜鳞状细胞癌单克隆细胞系Rca-B的建立及其生物学特性研究

Establishment and identification of biological characteristics of rat monoclone cell line Rca-B

目的建立大鼠颊黏膜鳞癌单克隆细胞系,并观察其生物学特性,为口腔癌的研究提供大鼠来源的恶性肿瘤细胞系、移植瘤和转移动物模型。方法将4-硝基喹啉-1-氧化物诱发的 SD大鼠颊黏膜鳞癌组织标本进行体外传代培养,利用有限稀释法获得单克隆细胞。光学和电子显微镜观察该细胞的形态变化,细胞计数和四唑盐比色法观察细胞的增殖能力,染色体分析确定细胞核型特点,流式细胞仪检测其细胞周期;动物实验观察细胞裸鼠皮下接种成瘤率及实验性肺转移能力。结果成功得到单克隆细胞系,细胞形态和生物学观察结果表明,该细胞系符合鳞癌细胞的基本特征,65代细胞群体倍增时间为25.44 h,平板克隆形成率为56.30%。细胞周期分布 S 期占20.13%;染色体为四倍体核型。角质蛋白和波形蛋白免疫组织化学染色均为阳性。细胞系裸鼠皮下接种成瘤为16/16;细胞系行裸鼠尾静脉注射后,其实验性肺转移为10/10;该单克隆细胞系 SD 大鼠同种异体皮下接种不能成瘤。结论成功建立 SD 大鼠颊黏膜鳞癌细胞系 Rca-B,细胞系具有高转移鳞癌细胞的典型生物学特征;该细胞系是研究口腔鳞癌分子发生机制和防治策略的又一理想模型。

Objective To establish a monoclone cell line of squamous cell carcinoma（SCC） in rat buccal mucosa and to study its biological characteristics. Methods SCC in rat oral mucosa was induced by adding 4-nitroquinoline-1-oxide （4NQO） into the SD rats＇ drinking water, and the cancer cells were then cultured to obtain mixed cells in vitro. The mixed tumor cells were purified by mono cell cloning method. The biological characteristics of the cells were studied by microscope and electronic microscope observation, chromosome analysis, Methyl thiazolyl tetrazolium （ MTT ） test, flow cytometry assay and immunohistochemistry staining. Hypodermic inoculations of the cells in nude mice and injection of the cells by nude mice tail veins were performed to observe the tumor formation and long distance metastasis. Results The morphology proved that the cell line was squamous cell carcinoma cells, which were cultured from one cell. The population doubling time for passage 65 cells was 25.44 hours. The cells in S-phase accounted for 20. 13% of the cell cycle. The chromosome modal number was 84. All the cells expressed the proteins of cytokeratin and vimentin. The xenograft rate and the tumor metastatic rate to the lung were 100% in nu/nu BALB/C mice, but the homograft rate was zero in SD Rats. Conclusions Rca-B was a typical oral squamous cell carcinoma cell line derived from Sprague-Dawley rat buccal mucosa carcinoma, and the cell line has high metastatic potential and its biological characteristics were well ascertained.