用原核增强子提高hTERT启动子介导的基因表达

Incorporation of prokaryotic enhancer for enhancement of gene expression driven by hTERT promoter

目的：探讨原核增强子能否提高hTERT启动子的转录活性及对其肿瘤特异性的影响。方法：将增强子SV40、CMV分别或组合构建一系列GFP和荧光素酶报告基因载体，转染人肝癌、鼻咽癌、宫颈癌、结直肠癌细胞、成纤维肉瘤和正常成纤维细胞，流式细胞仪计数和双荧光酶分析系统检测基因的表达效率。结果：在正常成纤维细胞中这些载体均不能有效表达；而在肿瘤细胞中hTERT启动子能介导基因表达但效率不高，增强子能使基因表达增强3～26倍，特别是单独CMV增强子和SV40-CMV双增强子使荧光素酶表达提高20-26倍，是常用的CMV增强子／启动子的2～7倍；完整CMV增强子／启动子对hTERT启动子的活性影响因不同细胞而异。结论：增强子能显著增强hTERT启动子的转录活性并对其肿瘤特异性没有影响，CMV增强子或SV40-CMV双增强子／hTERT启动子是高效特异的通用调控元件，用于靶向性肿瘤基因治疗具有巨大潜力。

OBJECTIVE： To investigate the effect of prokaryotic enhancers on transgene expression driven by hTERT promoter and on the specificity of the tumor-specific promoter. METHODS： Six transcriptional regulatory elements were constructed by appending a CMV or SV40 enhancer or CMV enhancer/promoter 5＇ to the hTERT promoter. Plasmids expressing luciferase or GFP from the series of regulatory elements were transfected into nasopharyngeal carcinoma, cervix cancer, fibrosarcoma, colorectal cancer and human diploid fibroblasts cell lines. RESULTS： When transfected into HDF cell, these plasmids gave similar low levels of expression. By contrast, gene expression of both reporters from the hTERT promoter was obsewed in the cancer cell lines and the presence of enhancer sequence gave 3 to 26-fold higher levels of luciferase expression than those from the hTERT promoter alone. In particular, in CNE2, Hela and BEI.7402 cells, the element with CMV enhancer or SV40-CMV double enhancer provided luciferase expression levels of 20-to 26-fold and 2-to 7-fold higher than those from two baseline constructs containing the hTERT promoter alone and the CMV enhancer/promoter, respectively. CONCLUSIONS： The selective and incresed gene expression controlled by these hybrid transcriptional regulatory elements is identified in the telomerase positive cancer cell lines. The element including CMV enhancer or SV40-CMV double enhancer/hTERT promoter as an effective and specific transcriptional regulatory element might be used for targeted cancer gene therapy.