T细胞识别的黑色素瘤抗原1在人脉络膜黑色素瘤细胞系中的表达

摘要

目的 观察T细胞识别的黑色素瘤抗原1 (MART-1)在人脉络膜黑色素瘤细胞系中的表达及启动子甲基化对其表达的可能影响.方法 人脉络膜黑色素瘤细胞系92-1、92-2、Me1285和Ocm3常规培养.采用免疫荧光染色流式细胞仪及Western blot检测各细胞系MART-1蛋白的表达;RT-PCR检测各细胞系中MART-1 mRNA转录水平;甲基敏感的限制性核酸内切酶消化结合Southern blot分析MART-1启动子DNA的甲基化状态.结果 免疫荧光染色流式细胞仪及Western blot检测结果显示,MART-1蛋白在92-1、92-2和Ocm3细胞系表达阳性,Me1285细胞系表达阴性.与蛋白检测结果一致,RT-PCR检测可见MART-1 mRNA在92-1、92-2、Ocm3细胞系中转录,Me1285未观察到转录.MART-1阳性细胞系92-1、92-2、Ocm3和阴性细胞系Me1285存在不同的甲基化模式.阳性细胞系在Msp Ⅰ/HpaⅡ位点甲基化,内含子Nru Ⅰ位点未甲基化;而阴性细胞系在Msp Ⅰ/HpaⅡ位点未甲基化,Nru Ⅰ位点过甲基化.结论 MART-1可在人脉络膜黑色素瘤细胞系92-1、92-2和Ocm3中表达.MART-1启动子甲基化模式的改变可能影响MART-1的转录.%Objective To observe the expression and transcription of MART-1 in human uveal melanoma cell lines 92-1,92-2,Ocm3,Me1285,as well as the possible effect ofmethylation on its expression.Methods The cell lines 92-1,92-2,Ocm3 and Me1285 were cultured routinely and tested for MART-1 expression at protein and mRNA level by FACS analysis,Western blot and RT-PCR respectively.Methylation status of the MART-1 promoter region in all the cell lines were checked by Southern blots of DNA digested with methylation sensitive restriction enzymes.Results As observed in FACS analysis and Western blot,92-1,92-2 and Ocm3 were MART-1 positive cell lines while Me1285 was negative cell line.Consistent with protein analysis,92-1 and Ocm3 cell lines showed MART-1 specific PCR products and there was no product in Me1285 cell line in RT-PCR.The MART-1 positive cell lines,92-1,92-2,and Ocm3 show methylation at the MspⅠ/Hpa Ⅱ site,and the Nru Ⅰ sites of all positive cell lines are not methylated.The MART-1 negative cell line Me1285 shows hyperrnethylation at the Nru Ⅰ site and the Msp Ⅰ/Hpa Ⅱ site is not methylated.Conclusions MART-1 could be expressed in human uveal melanoma cell lines 92-1,92-2 and Ocm3.The change of methylation status of MART-1 promoter may correlate with the transcription of MART-1.