中华皮肤科杂志 ›› 2006, Vol. 39 ›› Issue (4): 193-196.

• 论著 • 上一篇    下一篇

SV40T抗原基因转染正常人黑素细胞的研究

王鹰, 邓军, 杨希川, 郝飞, 钟白玉, 叶庆佾   

  1. 第三军医大学西南医院皮肤科 重庆 400038
  • 收稿日期:2005-08-31 发布日期:2006-04-15
  • 通讯作者: 郝飞,email:haofei@mail.tmmu.com.cn E-mail:haofei@mail.tmmu.com.cn
  • 基金资助:
    国家自然科学基金资助项目(30371299)

Transfection of normal human melanocytes with SV40 T antigen gene

WANG Ying, DENG Jun, YANG Xi-chuan, HAO Fei, ZHONG Bai-yu, YE Qing-yi   

  1. Dermatology Department of Southwest Hospital, Third Military Medical University, Chongqing 400038, China
  • Received:2005-08-31 Published:2006-04-15

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摘要： 目的 研究SV40T抗原基因对体外培养的人正常黑素细胞的转化作用。方法 用阳离子聚合物SofastTM介导基因转染,将含有SV40T抗原基因的真核表达载体SV40T-pEGFP导入原代培养的正常人黑素细胞进行稳定表达,G418筛选出阳性克隆,挑独立的单克隆扩大培养,检测SV40T抗原基因及SV40T蛋白在黑素细胞内的表达。结果 分离获得转化细胞阳性克隆。提取DNA及RNA后用PCR法成功扩增出288bp的片段,免疫组化及蛋白质印迹结果证实,转染黑素细胞核内有SV40T蛋白表达。结论 SV40T抗原基因可以成功诱导人黑素细胞的转化。

关键词: 黑素细胞, 猴病毒40, 抗原,病毒,肿瘤

Abstract: Objective To investigate the immortalization of human melanocytes by transfection with SV40 T antigen (SV40T). Methods By using SofastTM,a gene transfection reagent, the reconstructed eukaryotic expression vector SV40T-pEGFP was stably transfected into cultured primary human melanocytes, then the positive cells were selected with G418. After the positive cells were expanded in culture, the expression of SV40T gene was detected by RT-PCR and PCR, and the protein expression of SV40T by Western blotting. Results The genome DNA and total RNA were isolated from the positive cell clones, and a 288 bp fragment, which was specific for the SV40T antigen gene, was amplified. The results of immunohis-tochemistry and Western blotting confirmed the expression of SV40T protein in transfected cells. Conclusion SV40T antigen gene can successfully induce the immortalization of human melanocytes.

Key words: Melanocytes, Simian virus 40, Antigens, viral, tumor