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目的:构建融合启动子E6.hTERTp,以探讨放射干预后放射敏感启动子片段E6对人端粒酶逆转录酶启动子(hTERTp)启动活性以及对鼻咽癌靶向调控的影响。方法:通过Overlap PCR法合成融合启动子E6.hTERTp,连接至PGL3 basic质粒。构建含EGR-1启动子(EGR-1p)以及hTERTp的PGL3 basic载体做对照。使用Lipo2000将报告载体以及pRL-SV40质粒共转染正常人成纤维细胞HDF以及人鼻咽癌CNE-2细胞,并给予不同剂量放射干预,双荧光素酶法检测其启动活性。t检验分析不同细胞系中不同放射条件下各启动子启动活性差别。结果:在正常HDF细胞系内,无论放射与否,hTERTp组以及E6.hTERTp组的启动活性均很低;而在CNE2细胞系中,三种启动子启动活性均随放射剂量增加而增加,其增长幅度从大到小分别是EGR-1p组、E6.hTERTp组、hTERTp组。在相同放射剂量干预下,EGR-1P组、E6.hTERTp组与hTERTp组三组间的启动活性两两之间均有统计学差别(p<0.01)。结论:放射诱导型增强子E6能够明显提高hTERTp放射敏感性,且不影响其在鼻咽癌中靶向性调控作用,为该类肿瘤的放射-基因治疗提供了新的思路。
OBJECTIVE: To construct the fusion promoter E6.hTERTp to investigate the promoter activity of human telomerase reverse transcriptase promoter (hTERTp) and its effect on the target regulation of nasopharyngeal carcinoma after radioactive intervention. Methods: Fusion promoter E6.hTERTp was synthesized by Overlap PCR and ligated into PGL3 basic plasmid. The PGL3 basic vector containing the EGR-1 promoter (EGR-1p) and hTERTp was constructed as a control. The reporter vector and pRL-SV40 plasmid were co-transfected into HDF and human nasopharyngeal carcinoma CNE-2 cells by using Lipo2000. The cells were exposed to different doses of radioimmunoassay. The activation activity was detected by dual luciferase assay. t test analysis of different cell lines under different radiation conditions promoter promoter activity difference. RESULTS: In normal HDF cell lines, hTERTp group and E6.hTERTp group had low activation activity regardless of radioactivity or not. In CNE2 cell line, the promoter activity of all three promoters increased with increasing doses, Increasing from largest to smallest were EGR-1p group, E6.hTERTp group, hTERTp group. In the same dose of radiation intervention, EGR-1P group, E6.hTERTp group and hTERTp group of start-up activity between the two groups were statistically significant differences (p <0.01). CONCLUSION: E6, a radiation-inducible enhancer, can significantly enhance the radiosensitivity of hTERTp without affecting its target regulation in nasopharyngeal carcinoma, providing a new idea for radiation-gene therapy of this kind of tumor.