hTERT及CMV双调控TK基因靶向杀灭鼻咽癌细胞的实验研究

Targeted TK suicide gene vector regulated by two hTERT promoter and CMV enhancer for nasopharyngeal carcinoma targeted gene therapy

目的探讨hTERT启动子及CMV增强子双调控表达载体调控TK基因靶向杀灭鼻咽癌细胞的效应。方法将双调控增强型载体及hTERT单启动子载体(作为对照组)分别转染两种端粒酶(+)的人鼻咽癌5-8F细胞及对照组人乳腺癌MCF-7细胞及端粒酶(-)的正常人血管内皮ECV细胞,StretchPCR法验证5-8F细胞、MCF-7细胞及ECV细胞中端粒酶活性,荧光显微镜下观察TK基因绿色荧光蛋白表达差异,MTT法分析杀灭鼻咽癌细胞的效果。结果①Stretch PCR法验证人鼻咽癌细胞5-8F、人乳腺癌细胞MCF-7端粒酶活性呈阳性,人血管内皮ECV细胞端粒酶活性呈阴性。②增强型表达载体转染上述两种端粒酶(+)肿瘤细胞均有很强的荧光表达及TK基因的mRNA表达,单启动子亦有较强的荧光表达,但比增强型表达载体要弱,而ECV细胞几乎无绿色荧光。③加入GCV后,增强型表达载体对两种端粒酶(+)肿瘤细胞体外增殖均有明显抑制作用,且均高于单启动子组、空载体组及空白对照组,而增强型表达载体转染ECV细胞体外增殖无明显抑制作用。结论以hTERT启动子及CMV增强子双调控TK基因的表达载体能够靶向杀灭鼻咽癌细胞,且作用明显强于单启动子载体组。这种新型高效、靶向增强型载体有可能成为一种鼻咽癌临床靶向基因治疗的新策略。

Objective To explore targeted TK vector reconstruction regulated by two hTERT promoter CMV enhancer and its TK gene killing effect in nasopharyngeal carcinoma cells.MethodsThe enhancer TK vector was transfected into telomerase （＋） human 5-8 F cells of nasopharyngeal carcinoma, telomerase （＋） human MCF-7 cells of breast cancer and telomerase （-） human vascular endothelial ECV cells （the two latters as controls） respectively （being mono-promoter vector as control group）, and then telomerase activity was firstly determined with Stretch PCR; the expressions of TK gene green fluorescent protein were observed under fluorescence microscope and the quantitative expression difference of TK gene mRNA was detected with real-time fluorescent quantitative PCR in transfected tumour cells and ECV cells, and finally the killing effects to two human 5～8 F cells and MCF-7 cells of cancers were analyzed with MTT. The contents above were been as test indexes for the killing effect of bi-regulated TK vector. ResultsTelomerase activity in human NPC 5-8F cells and breast cancer MCF-7 cells was masculine,and was negative in human blood vessel endothelium ECV cells. A strong TK gene fluorescent show and TK mRNA expression were displayed after bi-regulated suicide gene vector was transfected into nasopharyngeal carcinoma 5-8 F cell lines and human breast cancer MCF-7 cell line, which is more stronger than those of mono-promoter group, and ECV cells transfected by the TK vector. Added GCV, a obvious tumour cells growth inhibition induced by enhanced vector was observed in nasopharyngeal carcinoma 5-8 F cell lines and human breast cancer MCF-7 cell line, which were higher than those of mono-promoter, and blank controls, but without depressant effect in ECV cells transfected by enhanced TK vector.ConclusionsTK vector regulated by two hTERT promoter and CMV enhancer can obviously kills specifically to the two tumour cells, and also there is a good specificity in the new vector. As a result, the new type, high performance and targeted enhanced TK gene vector may be a new tumour targeted gene therapy strategy clinically to aim directly at most manignent tumours including nasopharyngeal carcinoma with more extensive anti-cancer spectrum.