SV40TAg基因和Cre/loxP系统诱导正常人黑素细胞可逆性永生化的初探  

The reversible immortalization of human melanocytes mediated by SV40T antigen gene and Cre/loxP system

作　　者：王鹰[1] 邓军[1] 郝飞[1] 杨希川[1] 钟白玉[1] 

机构地区：[1]第三军医大学西南医院皮肤科,重庆400038

出　　处：《临床皮肤科杂志》2006年第10期627-630,共4页Journal of Clinical Dermatology

基　　金：国家自然科学基金资助项目(30371299)

摘　　要：目的:研究Cre/loxP系统和SV40TAg基因在体外对人正常黑素细胞的可逆性永生化诱导作用。方法:将Cre-ERT2反转录病毒导入包装细胞PA317,获得的病毒上清感染SV40TAg基因诱导转化的永生化黑素细胞,三苯氧胺(tamoxifen)诱导Cre重组酶表达,分别在第6天和第10天用PCR、反转录(RT)-PCR和免疫组化方法检测SV40TAg基因在感染病毒上清的永生化黑素细胞内的表达,并采用左旋多巴(L-Dopa)染色、透射电镜、软琼脂克隆试验等检测细胞的生物学性状和功能。结果:Cre重组酶反转录病毒上清感染永生化黑素细胞经嘌呤霉素筛选和三苯氧胺诱导Cre重组酶表达,第6天时可检测到感染细胞中有SV40TAg基因的表达,第10天后则在细胞中检测不到SV40TAg基因表达;L-Dopa染色、透射电镜等鉴定结果表明,感染细胞具有与原代黑素细胞相同的生物学性状和功能。结论:联合应用Cre/loxP系统和SV40TAg基因可以成功地诱导具有良好生物学安全性的人源性可逆性永生化黑素细胞。Objective： To study the reversible immortalization of human melanocytes mediated by SV40TAg and Cre/loxP sitespecific recombinase system. Methods： The retrovirus vector Cre-ERT2 was transfected package cell lines PA317, then the immortalized melanocytes transfected by SV40Tag was induced by the retrovirus supernatant and the cells were selected with puromycin. After that, Cre recombinase expression was induced with tamoxifen to excise SV40TAg gene. At the 6th and 10th days after Cre recombinase reaction, the expression of SV40TAg gene was detected using PCR, RT-PCR and immunohistochemical staining. Meanwhile, the cell biological characters were identified by using L-Dopa staining, transmission electron microscope and soft agar study. Results： Tamoxifen induced the Cre recombinase expression in infected cells which selected by puromycin. The PCR, RT-PCR and immunohistochemical staining results showed that at the 6th day after Cre recombinase action, there could detect SV40TAg gene expression in cells, but at 10th day there was no expression of SV40TAg gene. Identify results of cell biological characters showed that the infected cells had parental-melanocytes-like biological function. These cells could not survive under suspension condition in soft agar study. Conclusion： Human melanocytes can be mediated reversed immortalization with SV40TAg gene and Cre/loxP site-specific recombinase system.

关 键 词：CRE/LOXP系统 SV40T抗原 黑素细胞 永生化 可逆性 

分 类 号：R334.5[医药卫生—人体生理学] Q813.11[医药卫生—基础医学]