人端粒酶催化亚单位(hTERT)启动子的克隆及其在人肺癌细胞中转录活性的研究  

Cloning of human telomerase catalytic subunit gene promoter and studying on its specific transcriptional activities in human lung cancer cells

作　　者：唐小军 王艳萍[1] 周清[1] 朱文[1] 车国卫[1] 陈晓禾[1] 朱大兴[1] 孙芝琳[1] 

机构地区：[1]四川大学华西医院四川省肺癌分子重点实验室,成都610041

出　　处：《中国肺癌杂志》2004年第6期475-479,共5页Chinese Journal of Lung Cancer

基　　金：国家自然科学基金 (No .30 2 70 589)资助~~

摘　　要：目的　克隆人端粒酶催化亚单位 (hTERT)的启动子 ,并检测它在多种人肺癌细胞株和人胚肺成纤维细胞株中的转录活性 ,为肺癌靶向性基因治疗的研究奠定基础。方法　以人胚肾 2 93细胞基因组DNA为模板 ,应用PCR方法克隆hTERT 5′端上游旁侧序列长约 1.1kb的启动子片段 ,经DNA测序无误后克隆入荧光素酶报告质粒 pGL3 Basic的荧光素酶基因上游 ,构建 pGL3 hTERTp重组质粒 ,用脂质体法瞬时转染人肺癌细胞株A5 49、SPC A 1、LTEPa 2、NCI H44 6、YTMLC、GLC 82、A2 ,以及人胚肺成纤维细胞株MRC 5 ,转染 48h后检测hTERT启动子在各细胞株中的转录活性。结果　琼脂糖凝胶电泳显示PCR克隆的hTERT启动子片段长约 1.1kb。DNA测序结果与GenBank中hTERT启动子DNA序列完全一致 ,其 5′端和 3′端分别位于hTERT基因转录起始位点上游 112 6bp和 43bp ,片段长度为 10 84bp。采用双酶切和PCR两种方法鉴定 pGL3 hTERTp重组质粒 ,均显示构建成功。瞬时转染及荧光素酶活性检测实验显示 ,hTERT启动子在所检测的肺癌细胞株中均有高低不同的转录活性 ,而在MRC 5细胞株中无转录活性。结论　该实验克隆的 10 84bp大小的hTERT启动子在多种肺癌细胞株中均有转录活性 ,在人胚肺成纤维细胞中无转录活性。Objective To clone DNA sequence of human telomerase reverse transcriptase (hTERT) promoter, and study its transcriptional activities in various human lung cancer cells and normal lung cell. Methods hTERT promoter of 1.1 kb was amplified with polymerase chain reaction (PCR) method, utilizing DNA of 293 cell as template. After DNA sequencing with correct result, the hTERT promoter was inserted into luci-ferase reporter vectors (pGL3-Basic) to reconstruct a recombinant plasmid named pGL3-hTERTp. Then the reconstructed plasmid was transiently transfected into lung cancer cell lines A549, SPC-A-1, LTEPa-2, NCI-H446, YTMLC, GLC-82, A2 and human embryonic pulmonary fibroblast cell line MRC-5. The transcriptional activities of hTERT promoter in various cells were determined by measuring the luciferase activities after 48 hours of transfection. Results Electrophoresis demonstrated that cloned hTERT promoter was about 1.1 kb, and DNA sequencing showed a same sequence as registered in GenBank. The cloned hTERT promoter was located between 1 126 bp and 43 bp in upstream of the transcription start site ATG and the length was 1 086 bp. The recombinant plasmid pGL3-hTERTp was confirmed by double digestion and PCR method with correct results. Luciferase assay showed there were different transcriptional activities of hTERTp in various lung cancer cell lines, but not in the MRC-5 cell line. Conclusion The hTERT promoter cloned in this study has transcriptional activities in various lung cancer cell lines but not in normal cell. It may act as control element in tumor-targeting gene therapy with hTERT.

关 键 词：肺肿瘤 端粒酶催化亚单位(hTERT) 启动子 转录活性 

分 类 号：R734.2[医药卫生—肿瘤]