鼻咽癌细胞中增强型表达载体调控TK基因与端粒酶活性关系的研究

Study on the relationship between TK gene regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells

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 explore the relationship between TK gene expression regulated by enhanced suicide gene vector and telomerase activity in nasopharyngeal carcinoma cells.Method：The reformed reconstructed enhanced vector,pGL3-basic-EGFP-TK-hTRETp-CMV enhancer,and hTERT mono-promoter vector,pGL3-basic-EGFP-TK-hTRETp（as controls）,were transfected into telomerase（＋） nasopharyngeal carcinoma 5-8F cell lines,telomerase（＋） human breast cancer MCF-7 cell lines and telomerase（-） normal vascular endothelium cell lines respectively. TK gene green fluorescent protein was observed by fluorescence microscope. The expression of TK gene mRNA was measured by the real-time fluorescent quantified PCR and the telomerase activity was determined by the method of TRAP argentation in maligment tumour cells pre-and post-transfected by enhanced vector. Meanwhile the relationship beteewn TK and telomerase was analyzed.Result：①A strong TK gene fluorescent show and TK mRNA expression were displayed after the enhanced suicide gene vector was transfected into nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line,which were more stronger than those of mono-promoter group,pGL3-basic-EGFP-TK-hTRETp,and ECV cells transfected by enhanced suicide gene vector. Meanwhile,real-time fluorescent quantified PCR showed that the A value of enhanced vector group was higher than that of controls. ②Telomerase activity after transfection of enhanced vector and GCV was lower than those before by the method of TRAP argentation in nasopharyngeal carcinoma cell lines,but no change in normal control cells after transfection of enhanced vector and GCV.③ After adding GCV,the obvious inhibitory effect of tumour cells growth induced by pGL3-basic-EGFP-TK-hTRETp-CMV enhancer were observed in nasopharyngeal carcinoma 5-8F cell lines and human breast cancer MCF-7 cell line,which was higher than those of mono-promoter,pGL3-basic-EGFP-TK-hTRETp,pGL3-basic-EGFP3 and blank controls,but without inhibitory effect in ECV cells transfected by enhanced vector. Conclusion：TK gene expression is regulated by hTERT promoter and CMV enhancer,and then the telomerase activity is reduced and the cancer cells are specifically killed.But it is unclear how the telomerase are down-regulated by TK gene.