人端粒酶反转录酶启动子的克隆与肿瘤靶向治疗的研究

南方医科大学学报 ›› 2005, Vol. 25 ›› Issue (02): 207-211.

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人端粒酶反转录酶启动子的克隆与肿瘤靶向治疗的研究

李帆¹, 谭晓华², 彭晓君¹, 蔡红兵³, 胡大荣¹

1. 北京军区总医院肝病治疗中心, 北京, 100700;
2. 北京军区总医院肿瘤诊疗中心, 北京, 100700;
3. 南方医科大学中医系, 广东, 广州, 510515

出版日期:2005-02-20 发布日期:2005-02-20
基金资助:
收稿日期:2004-8-3。
作者简介:李帆(1975- ),男,2004年毕业于解放军军医进修学院,硕士,医师,E-mail:fantist510@hotmail.com

Cloning of human telomerase reverse transcriptase promoter for targeted cancer therapy

LI Fan¹, TAN Xiao-hua², PENG Xiao-jun¹, CAI Hong-bing³, HU Da-rong¹

1. 北京军区总医院肝病治疗中心, 北京, 100700;
2. 北京军区总医院肿瘤诊疗中心, 北京, 100700;
3. 南方医科大学中医系, 广东, 广州, 510515

Online:2005-02-20 Published:2005-02-20

摘要/Abstract

摘要： 目的克隆hTERT启动子,并观察hTERT启动子调控下tk/GCV系统对人肺癌细胞系A549的特异性杀伤效果。方法设计特异性引物,以HepG2基因组DNA为模板,用PCR方法扩增hTERT启动子;分别构建hTERT启动子和hCMV启动子调控的LacZ、tk基因真核表达载体;将上述质粒用脂质体转染A549细胞和人胚肺成纤维细胞系MRC5后,通过RT-PCR和β-Gal染色观察hTERT启动子工作情况;用MTT法检测不同浓度的GCV对A549和MRC5细胞的细胞毒作用。结果成功克隆hTERT上游-280bp~+40bp的核心启动子。RT-PCR和β-Gal染色显示hTERT启动子能有效地调控其下游tk基因、LacZ基因在端粒酶阳性的肿瘤细胞中特异性转录和表达;hTERT启动子调控的tk/GCV系统对端粒酶阳性的肿瘤细胞系A549有明显的特异性杀伤效应。结论 hTERT启动子可特异性地调控目的基因在端粒酶阳性的肿瘤细胞中表达而不影响正常的细胞,表明端粒酶启动子调控自杀基因的表达是一种很有希望的肿瘤靶向基因治疗策略。

Abstract: Objective An human telomerase reverse transcriptase(hTERT) promoter was cloned to investigate the effect of simplex virus-thymidine kinase(HSV-tk) gene-ganciclovir(GCV) system under control of this promoter on lung cancer cells A549. Methods Specific primers were designed to amplify the core hTERT promoter from HepG2 genome. A set of expression vectors encoding LacZ or tk gene under control of hTERT or hCMV promoter was constructed through standard molecular cloning methods. After the transfection with lipofectamine 2000, reverse transcriptional PCR(RT-PCR) and β-Gal staining were performed to examine the activity of the hTERT promoter. The cytotoxic effects of GCV/tk on A549 and MRC5 cells transfected with the plasmids encoding tk gene were evaluated by MTT assay. Results The hTERT promoter containing-208-+40 bp of the upstream sequence of human telomerase was successfully cloned. β-Gal staining and RT-PCR were used to detect the expression of Lac Z and the transcription of tk gene under control of the hTERT promoter, respectively, in A549 rather than MRC5 cells. Cyototoxic effect of GCV was observed only in the A549 but not MRC5 cells after the transfection with pDC511 hTERT/tk, and the effect was dose-dependent. The effect, however, was observed in cells transfected with pDC518 hCMV/tk. Conclusion The hTERT promoter cloned in this study can specifically control the target gene expression in telomerase-positive tumor cells but not the normal cells, suggesting that the HSV-tk/GCV system under control of the hTERT promoter is a promising targeted gene therapy for malignant tumors.

中图分类号:

R731.31

李帆¹, 谭晓华², 彭晓君¹, 蔡红兵³, 胡大荣¹. 人端粒酶反转录酶启动子的克隆与肿瘤靶向治疗的研究[J]. 南方医科大学学报, 2005, 25(02): 207-211.

LI Fan¹, TAN Xiao-hua², PENG Xiao-jun¹, CAI Hong-bing³, HU Da-rong¹. Cloning of human telomerase reverse transcriptase promoter for targeted cancer therapy[J]. Journal of Southern Medical University, 2005, 25(02): 207-211.

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