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目的:探讨人端粒酶催化亚单位(hTERT)基因核心启动子和原核增强子CMV联合调控单纯疱疹病毒胸苷激酶基因/更昔洛韦系统对TK基因的活性改变及与鼻咽癌细胞中端粒酶活性的关系。方法:将改良构建后的增强型载体pGL3-basic-EGFP-TK-hTRETp-CMV enhancer及单启动子载体pGL3-basic-EGFP-TK-hTRETp(作为对照组)分别转染端粒酶阳性的人鼻咽癌5-8F细胞株及对照组细胞人乳腺癌MCF-7细胞(端粒酶阳性)及正常人血管内皮ECV细胞(端粒酶阴性),采用荧光显微镜下观察其TK基因绿色荧光蛋白表达,实时荧光定量PCR方法检测转染细胞中TK基因mRNA定量表达差异,TRAP银染法检测肿瘤细胞转染前后端粒酶活性改变并分析TK基因表达与端粒酶活性之间的关系。结果:①增强型表达载体转染鼻咽癌5-8F细胞及乳腺癌MCF-7细胞均有很强的荧光表达及TK基因的mRNA表达,比单启动子pGL3-basic-EGFP-TK-hTRETp及ECV细胞绿色荧光要强。实时荧光定量PCR显示,增强型载体组A值亦较对照组明显增高。②转染增强型载体(加GCV)后TRAP银染法检测鼻咽癌5-8F细胞端粒酶活性较转染前明显降低,但转染正常对照细胞后活性无变化。③加入GCV后,pGL3-basic-EGFP-TK-hTRETp-CMV enhancer对鼻咽癌5-8F细胞及乳腺癌MCF-7细胞体外增殖均有明显抑制作用,高于单启动子组pGL3-basic-EGFP-TK-hTRETp及空载体组pGL3-basic-EG-FP3及空白对照组,而pGL3-basic-EGFP-TK-hTRETp-CMV enhancer转染ECV细胞无明显抑制作用。结论:hTERT启动子及CMV增强子可明显增强TK基因活性,并可导致该种肿瘤细胞端粒酶活性降低,靶向杀灭该种肿瘤细胞,但这种由TK基因介导的端粒酶活性抑制机制尚不清楚。
OBJECTIVE: To investigate the effect of hTERT gene core promoter and prokaryotic enhancer (CMV) in regulating the activity of TK gene in herpes simplex virus thymidine kinase gene / ganciclovir system and in nasopharyngeal carcinoma cells Relationship between telomerase activity. Methods: The telomerase positive individuals were transfected with pGL3-basic-EGFP-TK-hTRETp-CMV enhancer and single promoter pGL3-basic-EGFP-TK-hTRETp Nasopharyngeal carcinoma cell line 5-8F and control group, human breast cancer MCF-7 cells (telomerase positive) and normal human vascular endothelial ECV cells (telomerase negative), observed under a fluorescent microscope its TK gene green fluorescent protein The expression of TK gene mRNA in transfected cells was detected by real-time fluorescence quantitative PCR. The changes of telomerase activity before and after transfection were detected by TRAP silver staining. The relationship between TK gene expression and telomerase activity was analyzed. Results: (1) The expression of TK gene and the mRNA of the transfected nasopharyngeal carcinoma cell line 5-8F and the breast cancer cell line MCF-7 were significantly enhanced compared with the single promoter pGL3-basic-EGFP-TK-hTRETp And ECV cells stronger green fluorescence. Real-time PCR showed that the A value of enhanced vector group was significantly higher than that of the control group. ② The transfection of enhanced vector (plus GCV) TRAP silver staining assay nasopharyngeal carcinoma 5-8F cell telomerase activity was significantly lower than before transfection, but the transfected cells did not change the activity of normal control. ③ After adding GCV, pGL3-basic-EGFP-TK-hTRETp-CMV enhancer significantly inhibited the proliferation of nasopharyngeal carcinoma 5-8F cells and breast cancer MCF-7 cells in vitro, which was higher than that of pGL3-basic- EGFP-TK-hTRETp, pGL3-basic-EG-FP3 and blank control group, while pGL3-basic-EGFP-TK-hTRETp-CMV enhancer had no obvious inhibitory effect on ECV cells. Conclusion: hTERT promoter and CMV enhancer can significantly enhance the activity of TK gene and lead to the decrease of telomerase activity in this kind of tumor cells, targeting to kill this kind of tumor cells. However, this TK gene-mediated telomerase The mechanism of activity inhibition is not clear.