靶向增强型TK表达载体在鼻咽癌细胞中的作用研究

Effects of target-enhanced TK gene vector in nasopharyngeal carcinoma cells

目的探讨hTERT启动子及CMV增强子双调控机制的增强型表达载体pGL3-bas-ic-EGFP-TK-hTERTp-CMV enhancer在鼻咽癌细胞中的靶向杀伤效应。方法构建靶向性增强型hTERT启动子及CMV增强子双调控TK表达载体pGL3-basic-EGFP-TK-hTRETp-CMV enhancer,并以单启动子载体pGL3-basic-EGFP-TK-hTRETp作为对照组,分别转染端粒酶阳性的人鼻咽癌细胞株5-8F、人乳腺癌细胞MCF-7(阳性对照)及正常人血管内皮细胞ECV(阴性对照)。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,Stretch PCR法检测肿瘤细胞端粒酶活性,MTT法分析杀灭鼻咽癌细胞的效果等作为检验指标。结果①该增强型表达载体转染5-8F细胞及MCF-7细胞后的绿色荧光表达及TK基因的mRNA表达均强于单启动子pGL3-basic-EGFP-TK-hTRETp组;而ECV细胞只有极微弱的绿色荧光和极弱的TK基因的mRNA表达。实时荧光定量PCR显示,增强型载体组A值较对照组单启动子组明显增高,是单启动子组的2～5倍。StretchPCR法检测转染前后5-8F细胞端粒酶活性明显被抑制。②加入GCV后增强载体组对鼻咽癌5-8F细胞及乳腺癌MCF-7细胞体外增殖均有明显抑制作用,均高于单启动子载体组、不加GCV的增强型载体组、空载体组及空白对照组。结论以hTERT启动子及CMV增强子双调控机制介导TK基因的新型靶向增强型表达载体能够高效靶向性杀灭鼻咽癌细胞,这种新型高效靶向增强型载体有可能成为一种针对包括鼻咽癌在内的具有较为广泛抗癌谱的恶性肿瘤临床靶向基因治疗的新策略。

Objective To explore the killing effect of enhanced TK vector regulated by hTERT promoter and CMV enhancer,pGL3-basic-EGFP-TK-hTRETp-CMV enhancer,in nasopharyngeal carcinoma cells. Methods pGL3-basic as a basic vector template,a target-enhanced TK vector,pGL3-basic-EGFP-TK-hTRETp-CMV enhancer,was cut,linked,and constructed by restriction enzymes （regulated by hTERT promoter and CMV enhancer）. Mono-promoter vector,pGL3-basic-EGFP-TK -hTRETp,was chosen as control group; the vectors were transfected into telomerase （＋） 5-8F cells of human nasopharyngeal carcinoma and telomerase （＋） human MCF-7 cells of breast cancer （positve control） and telomerase （-） human vascular endothelial ECV cells （negative control） respectively. The expression of TK gene green fluorescent protein was observed under fluorescence microscope,the difference of quantitative expression of TK gene mRNA was detected with real-time fluorescent quantitative PCR; telomerase activity was determined with Stretch PCR in maligment tumour cells,and the killing effects of the vectors to the 5-8F cells and MCF-7 cells were analyzed with MTT. The above-mentioned indexes were chosen to evaluate the killing effects of enhanced TK vector. Results ① Strong expression of TK gene green fluorescent protein and TK mRNA was displayed in enhanced-vector transfected 5-8F cell line and MCF-7 cell line,while that in the mono-promoter transfected cell lines and ECV cells transfected by enhanced TK vector was weak. Real-time fluorescent quantative PCR also showed that the A-value of enhanced TK vector group was higher than that of the control group. Stretch PCR showed that the telomerase activity in the 5-8F cell line transfected by enhanced TK vector was inhibited. ② After adding GCV,obvious cell growth inhibition was observed in pGL3-basic-EGFP-TK-hTRETp-CMV enhancer transfected 5-8F cell line and MCF-7 cell line,which was more obvious than those of pGL3-basic-EGFP-TK-hTRETp,pGL3-basic-EGFP-TK-hTRETp-CMV without GCV group,pGL3-basic-EGFP group and the blank control. Conclusion Target-enhanced TK vector regulated by hTERT promoter and CMV enhancer can effectively kill 5-8F cells of human nasopharyngeal carcinoma with good specificity,which indicates this target-enhanced TK gene vector may be useful in the strategy of target gene therapy of malignant tumours including nasopharyngeal carcinoma.