SV40 T基因诱导的人卵巢癌细胞永生化

Immortalization of human ovarian carcinoma cells induced by SV40 T antigen gene

目的 建立人卵巢癌永生化细胞系 ,为卵巢癌体外研究提供实验模型。方法 利用基因重组技术构建猴肾病毒 4 0 (SV4 0 )T抗原基因的高效真核表达载体 pcD2 SV4 0。将其转染 10例原代培养的早期传代细胞 ,经G4 18筛选 ,抗性克隆扩大培养 ,建立永生化细胞系。免疫组化检测细胞的上皮起源 ,用PCR、RT PCR和SouthernBlot方法鉴定SV4 0T基因在转染细胞中的表达。观察所建立的永生化细胞系的染色体核型。结果 10例转染细胞中 ,6例筛选出抗性克隆。角蛋白免疫组化证实 3例为上皮起源。其中 2例上皮起源细胞得以扩大培养成系 ,长期稳定传代 ,分别命名为BUPH :OVSC 2和BUPH :OVCA 3。经鉴定两种细胞系中存在SV4 0T在基因水平及转录水平的表达。其染色体核型均符合人体恶性细胞特征。结论 利用SV4 0T抗原基因进行人卵巢癌原代细胞的永生化是一种可行的、可重复的建系方法 ,可提高卵巢癌细胞成系机率。

Objective To establish immortalized human ovarian carcinoma cell lines and provide experimental models for the investigation of ovarian carcinoma. Methods An eukaryotic vector expressing SV40 T antigen gene (pcD2 SV40) was constructed by DNA recombinant technique. 10 cases of primary human ovarian carcinoma cells were cultured and transfected with plasmid pcD2 SV40. Colonies were isolated by G418 selection and expanded to immortalized cell lines. Their epithelial origins were detected by the present of cytokeratin by immunohistochemistry method. The expression of SV40 T gene in expanded cell lines was identified by PCR, RT PCR and Southern Blot. The karyotype analyses of cell lines were observed. Results Among 10 cases, resistant colonies were isolated in 6 cases. Epithelial origin was demonstrated in 3 cases,2 of them were expanded to immortalized cell lines, named as BUPH:OVSC 2 and BUPH:OVCA 3 respectively. Stable growth and serial propagation had been observed in the two cell lines. Also the expression of SV40 T gene in the cell lines was demonstrated in both gene level and transcriptional level. The karyotype analyses exposed their malignant characteristics. Conclusion SV40-medicated immortalization of primary human ovarian carcinoma cells is a useful and reproducible method to acquire continuous cell supply of different types of ovarian carcinoma for investigation.