放射诱导型增强子E6可提高hTERTp的放射敏感性

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。

The radiosensitivity of hTERT promoter enhanced by radiosensitive enhancer E6

Objective： Construct novel promotor E6.hTERTp to detect the influence of radiosensitive enhancer E6 to hTERTp＇s targeting promotive effect. Methods： The fusion gene E6.hTERTp was constructed by overlap PCR and was inserted into PGL3 basic plasmid. PGL3-hTERTp and pGL3-EGRlp were also constructed for comparison. All the plasmids together with pRL-SV40 plasmids were transfected into CNE-2 cells and HDF cells with liposome .The promotive effect of recombinant promoter E6.hTERTp in CNE-2 cells and HDF cells were measured by dual luciferase reporter gene assay system and was compared with EGR-Ip and hTERTp for difference between every two groups under different dose of radiation .The results were analyzed with t test; Results： The promotive effects of hTERT promoter and E6.hTERTp were absent in HDF cells with any dose of radiation; While in CNE-2 cells, increase of promotive effects in accordance with the radiation dose could be observed in all groups. The change to baseline （0Gy） in descending sequence were the EGR-1p group; the E6.hTERTp group and the hTERTp group; under the same dose of radiation, the difference between the promo- tive effect of EGR-1p, E6.hTERTp group and hTERTp group were statistically significant（p〈0.01 ）. Conclusion： Radiosensitive enhancer E6 increases the radiosensitive promotive effect of hTERTp in CNE-2 cells and does not decrease its NPC targeting character. This can be a potential strategy of radio-gene therapy for tumors mainly treated by radiation.