摘要
目的:比较不同长度的hTERT启动子以及Survivin启动子在肺癌A549细胞中的启动活性,为肺癌靶向性基因治疗提供依据。方法:用PCR法扩增1084 bp hTERT启动子、980 bp Survivin启动子和红色荧光蛋白基因;在真核表达质粒pGL3-hTERT基础上分别构建3种启动子调控的、以红色荧光蛋白CDS为目的基因的真核表达质粒pGL-H-RED、pGL-LH-RED和pGL-S-RED。将重组质粒用脂质体分别转染A549和MRC-5细胞,72h后用Imagepro-Plus6.0分析3种启动子在相同时间内启动红色荧光蛋白的表达水平。结果:重组质粒在A549细胞中观察到了红色荧光,在MRC-5细胞中无红色荧光。pGL-S-RED质粒在A549细胞中表达最强,pGL-LH-RED次之,pGL-H-RED最弱,3种质粒在A549细胞中的荧光强度(IOD)分别为201.17、171.70和136.34。结论:所克隆的hTERT启动子和Survivin启动子均表现出肿瘤特异性,其中Survivin启动子在肺癌A549细胞中具有最好的启动效应,可望开发成为肺癌靶向性基因治疗工具。
Objectives： To compare the transcriptional activities of tumor-specific hTERT promoters and survivin promoter in human lung cancer A549 cells, in order to lay some groundwork for targeting gene therapy in human lung cancer. Methods： The 1 084 bp fragment of hTERT promoter and 980bp survivin promoter from A549 cells were acquired by PCR amplification. The eukaryotic expression plasmids （pGL-H-RED, pGL-LH-RED and pGL-S-RED ） which have Red Fluorescin reporter gene （Red） and are modulated by 263 bp hTERT promoter, 1084bp hTERT promoter and 980bp survivin promoter respectively were constructed based on the pGL3-hTERT which has 263bp hTERT promoter from HepG2 cells. These recombinant plsmids were transfected into A549 and MRC-5 cells with liposome. After 72 hours, the expression level of red fluorescin target protein modulated by 3 promoters at the same time was analyzed by Imagepro-Plus6. 0 software. Results： The red fluorescin target protein was observed in transfected A549 cells,but there was no red fluorescence in transfected MRC-5 control cells. The pGL-S-RED had the highest expression activity in A549 cells and the strength rate（ IOD ） of red fluorescence of A549 cells transfected by pGL-S-RED, pGL-LH-RED and pGL-H-RED were 201.17,171.70, and 136.34 respectively. Conclusion： Our data reveals that the cloned hTERT and survivin promoter are tumor-specific, and the survivin promoter has the highest transcriptional activities in lung cancer A549 ceils, and may serve as a useful tool for transcriptional targeting gene therapy of human lung cancer.
出处
《重庆医科大学学报》
CAS
CSCD
北大核心
2009年第2期168-173,共6页
Journal of Chongqing Medical University
作者简介
李奕璇（1975-），女，硕士，研究方向：恶性肿瘤的靶向性基因治疗。
通讯作者：陈全，男，副教授，E—mail：quanchen@tom．com。
