SV40T基因转染黑素细胞遗传稳定性研究  

Genetic stability of immortal melanocytes transfected by SV40T antigen gene

作　　者：常海[1] 邓军[1] 杨希川[1] 郝飞[1] 钟白玉[1] 叶庆佾[1] 

机构地区：[1]第三军医大学附属西南医院皮肤性病科,重庆400038

出　　处：《实用医院临床杂志》2008年第3期24-26,共3页Practical Journal of Clinical Medicine

基　　金：国家自然科学基金资助项目(30371299)

摘　　要：目的探讨SV40T抗原基因转染构建的永生化黑素细胞的遗传稳定性。方法体外培养黑素细胞,对第20代的永生化黑素细胞进行四甲基偶氮唑蓝(MTT)比色法分析细胞增殖情况,同时分析染色体核型,流式细胞仪分析细胞周期和DNA倍体;逆转录-聚合酶链反应(RT-PCR)半定量分析第20代细胞hMLH1、hMSH2错配修复基因表达,并提取细胞基因组,微卫星位点PCR产物进行变性聚丙烯酰胺电泳分析微卫星不稳定性。结果经过连续培养的第20代转染的黑素细胞左旋多巴染色阳性,增殖速度较原代培养黑素细胞明显增快,细胞周期S+G2期细胞比例较原代黑素细胞增多,同时未见异常DNA倍体,染色体仍为2倍体,转染细胞的hMLH1、hMSH2错配修复基因表达较正常黑素细胞上调;在1p22(D1S187)、5q14(D5S107)、18q12.2-12.3(D18S34)3个位点未出现微卫星不稳定性(m icrosatellite instab ili-ty,M I)。结论SV40T抗原基因转染的黑素细胞体外培养不出现遗传稳定性的异常改变。Objective To investigate the genetic stability of immortal melanocytes mediated by transfecting SV40T antigen gene. Methods We studied growth of the 20th immortal melanocytes by MTT and genetic stability through analysing karyotype and cell cycle and DNA ploid with a flow cytometer. We also tested the expression of mRNA of hMLH1 and hMSH2 and two important mismatch repair genes（MMR） through semiquantitative analysis by RT-PCR and compared three microsatellites localized to specific chromosome regions ： lp22 （ D1S187 ）, 5q14 （ D5S107 ）, 18q12. 2-12.3 （ D18S34 ） in genome of the immortal melanocytes with one of the normal cells. Results The cells kept normal karyotype but grew faster than the normal melanoeytes and had a larger percentage of S ＋ G2 stages in cell cycle. Semiquantitative analysis showed mRNA of hMLH1 and hMSH2 being up-regulated compared to the normal cells but mierosatellite instability（MI） was not found. Conclusion The immortal melanoeytes transfeeted by SV40T antigen gene keep normal genetic stability.

关 键 词：SV40T基因 黑素细胞 错配修复基因 微卫星不稳定性 

分 类 号：R394.2[医药卫生—医学遗传学]