猿猴病毒40T抗原介导永生化黑素细胞对其核型、HLA-DR和hTERT表达的影响  

Effect of immortalized melanocyte transfected with SV40T gene on karyotype and expression of HLA-DR and hTERT

作　　者：常海[1] 邓军[1] 郝飞[1] 杨希川[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：《临床皮肤科杂志》2008年第6期366-369,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:观察猿猴病毒40T(SV40T)抗原介导黑素细胞(melanocyte,MC)永生化对染色体核型、人白细胞抗原-DR(humanleucocyte antigen-DR,HLA-DR)和人端粒酶反转录酶(human telomerase reverse transcriptase,hTERT)表达的影响。方法:利用SV40T抗原基因和真核表达载体PEGFP,经SofastTM阳离子聚合物转染至体外培养的第4代MC细胞,使其永生化,通过G418筛选,滤纸片法挑选单一克隆。对培养至第15代的永生化细胞进行染色体分析,免疫组化方法检测HLA-DR和hTERT蛋白的表达,反转录(RT)-PCR检测hTERT mRNA表达,以人黑素瘤A375细胞株为对照。结果:SV40T抗原介导的永生化黑素细胞具有正常的二倍体,HLA-DR和hTERT的表达未受影响,均为阴性,同时也未检测到hTERT mRNA的表达。结论:①转化的MC染色体为正常核型、HLA-DR的表达不受体外长期传代培养和SV40T抗原的影响;②SV40T抗原不通过上调端粒酶反转录酶活性途径介导细胞永生化,同时也说明该转化细胞不易向恶性转化。Objective： To observe the effect on karyotype and expression of HLA-DR and hTERT of melanocyte immortalized by SV40T gene. Methods： The vector SV40T-Pegfp was transfected into cultured human melanocytes in vitro by using SofastTM, a gene transfection reagent, and the positive cells were selected by G418. The gene and protein of SV40T in the positive cells were identified by PCR,RT-PCR and immunohistochemistry. The proteins of HLA-DR and hTERT expressed in the cells were investigated by immunohistochemistry, and mRNA of hTERT by RT-PCR as well. Results： Both the proteins of HLA-DR,hTERT and hTERT mRNA were not detected in the immortalized melanocytes of the 15th passage. The cells also retained the normal karyotype. Conclusions： （1）The karyotype and expression of HLA-DR did not change in the immortalized melanocytes. （2）The mechanism of immortalization of melanocytes was not passed through upregulating the expression of hTERT, it also indicated that the cells could not be easily transformed to malignancy.

关 键 词：黑素细胞 抗原 猿猴病毒40T 人类白细胞抗原DR 人端粒酶反转录酶 

分 类 号：Q786[生物学—分子生物学] Q813.11