胸苷激酶基因在人二倍体成纤维细胞衰老过程中的表达调控

Regulation Mechanism of TK Gene Expression in Cellular Senescence of Human Diploid Fibroblasts

为研究人胸苷激酶 (humanthymidinekinase ,hTK)基因在复制衰老细胞及早衰细胞中表达下调的分子机制 ,构建了含hTK启动子的荧光素酶报告基因载体 .转染结果显示 ,复制衰老细胞与早衰细胞中hTK启动子的转录活性比年轻细胞中下降了近 3倍 ,表明转录水平的调控是hTK在衰老细胞中表达下降的主要调控机制 .定点突变的结果显示 ,转录因子Sp1、NF Y结合位点的突变可使hTK启动子活性降低近 5 0 % ,而E2F结合位点的突变可使其活性升高 2倍多 ,提示Sp1和NF Y是hTK基因的转录活化因子 ,而E2F为转录抑制因子 .电泳迁移率变更实验发现 ,与年轻细胞相比 ,Sp1、NF Y与hTK启动子的DNA结合活性在复制衰老细胞和早衰细胞中无明显改变 ,提示转录活化因子Sp1、NF Y并非hTK在衰老细胞中下调的主要因素 .染色质免疫共沉淀结果显示 ,在细胞内Rb结合在hTK启动子上 ,且同年轻细胞相比 ,复制衰老细胞及早衰细胞中的hTK启动子结合着更多的Rb ,这提示细胞衰老过程中Rb的去磷酸化可能与hTK基因在衰老过程中的下调有关 .

In order to study the molecular mechanism of down regulation of human thymidine kinase(hTK) gene expression in replicative senescent and premature senescent human diploid fibroblasts (HDFs), luciferase reporter vectors containing hTK promoter fragment were consturcted. The results of transcription showed that the hTK promoter activity was about 3 fold higher in young HDFs than in replicative senescent and premature senescent HDFs, indicating that transcriptional regulation was involved in the down regulation of hTK expression in cellular senescence. The site-directed mutagenesis showed mutations at binding sites for transcriptional factors Sp1 and NF-Y both caused about 50% reduction in hTK promoter activity, whereas the mutations at E2F sites increased the promoter activity about 2 fold, suggesting that Sp1 and NF-Y were transcriptional activators for hTK, but E2F was a transcriptional repressor. The EMSA analysis indicated that the DNA binding activity of both Sp1 and NF-Y did not significantly change in replicative senescent and premature senescent HDFs compared with that in young HDFs, suggesting that transcriptional activators Sp1 and NF-Y did not play major roles in down regulation of hTK expression in cellular senescence. The chromatin immunoprecipitation (Chip) demonstrated for the first time that Rb binded to hTK promoter in HDFs, and in replicative senescent and premature senescent HDFs, there was more Rb binding to hTK promoter than in young HDFs, suggesting dephosphorylation of Rb in senescent cells may account for the down regulation of hTK in cellular senescence.