Construction of retroviral vector expressing wild type p53 gene driven by hTERT promoter firstly by retrovirus only infecting dividing cells

Abstract

AIM：To construct a vector expressing through target gene given by human telomerase reserve transcriptase (hTERT) promoter with pLHCX as vector and realize target expression.

METHODS：Experiments were performed at the Key Laboratory of Molecular Medicine from September 2005 to May 2006. ①The phTERT-luc plasmid containing 319 bp hTERT core promoter was presented by Dr. Zheng from Military Academy of Science; Retrovirus vector pLHCX was presented by Professor Zhu from Zhejiang University; The plasmid pCMV-Neo-BamH-wtp53 containing 1.8 kb wtp53 gene was presented by Professor Zhu from Second Military Medical University of Chinese PLA; Enhanced green fluorescent protein report plasmid pEGFP-N2 and pEGFP-C1 were kept in this laboratory; human carcinoma of large intestine cell strain SW620 and HT-29, human cervical carcinoma cell strain Hela, human lung carcinoma cell strain A549, and human lung embryo fibroblast MRC-5 were kept in this laboratory. ②The plasmid pLHCX-hTERT-EGFP expressing EGFP driven by hTERT promoter and the plasmid pLHCX-hTERT-wtp53 expressing wtp53 also driven by hTERT promoter were constructed through directional cloning. The plasmid pLHCX-hTERT-EGFP and the plasmid pLHCX-CMV-EGFP were used for the transfection of human carcinoma of large intestine cell strain SW620 and HT-29, human cervical carcinoma cell strain Hela, human lung carcinoma cell strain A549, and human lung embryo fibroblast MRC-5. ③The flow cytometer was used to analyze the transfection efficiency of pLHCX-hTERT-EGFP so as to declare the hTERT promoter specific expression in tumor cells. The human colon cancer cell line SW620, which the p53 gene is mutation, were infected with the plasmid pLHCX-hTERT-wtp53 and p53 mRNA, p53 protein were demonstrated with RT-PCR and Western blot. The normal human embryo lung fibroblast cell strain MRC-5 and the human colon cancer cell line SW620 and HT-29 were treated with the plasmid pLHCX-hTERT-wtp53, the propagate inhibition and apoptosis of cells were detected by MTT and FCM, respectively.

RESULTS: ①The plasmids pLHCX-hTERT-EGFP and pLHCX-hTERT-wtp53 were successfully constructed, which were confirmed by restriction endonuclease and sequencing. Green fluorescent protein expression was observed by pLHCX-hTERT-EGFP transfection SW620. The sequencing verified that complete wtp53 fragment was found. ②Flow cytometry declared that enhanced green fluorescent protein expression was significantly higher in the SW620, Hela and A549 than the MRC-5 after pLHCX-hTERT-EGFP treatment (P < 0.05), but no significant difference was found in positive and negative cells above mentioned (P > 0.05). ③The expression of wtp53 mRNA and the protein were demonstrated by RT-PCR and Western blot, respectively. ④MTT showed that recombinant plasmid had significant inhibition on carcinoma of large intestine with positive telomerase, but had no significant effects on MRC-5 cells with negative telomerase (P < 0.05). ⑤Flow cytometry showed that the apoptosis of carcinoma of large intestine with positive telomerase was significantly higher than the MRC-5 cells with negative telomerase induced by recombinant plasmid (P < 0.05).

CONCLUSION:The retroviral vector pLHCX-hTERT-EGFP can express in tumor cells specifically and the retroviral vector pLHCX-hTERT-wtp53 can affect carcinoma of large intestine with positive telomerase specifically.

Yu X, Hong K, Deng JH, Liu TD, Lei J, Zhang JX, Shao JH.Construction of retroviral vector expressing wild type p53 gene driven by hTERT promoter firstly by retrovirus only infecting dividing cells.Zhongguo Zuzhi Gongcheng Yanjiu yu Linchuang Kangfu 2008;12(3):493-497(China) [www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-493(ps).pdf]

摘要
目的：选择只感染分裂细胞的鼠源性反转录病毒载体pLHCX作为载体,构建由hTERT启动子驱动目的基因表达的载体，实现靶向性表达。
方法:实验于2005-09/2006-05在江西省分子医学重点实验室完成。①实验材料：含319 bp hTERT核心启动子的phTERT-luc质粒由军事科学院郑晓飞博士惠赠，反转录病毒载体质粒pLHCX由浙江大学朱永良教授惠赠，含1.8 kb wtp53基因的质粒pCMV-Neo-BamH-wtp53由第二军医大学朱明华教授惠赠，增强型绿色荧光蛋白报告质粒pEGFP-N2和pEGFP-C1由本室保存，对照反转录病毒质粒pLHCX-CMV-EGFP前期构建完成；端粒酶阳性人大肠癌细胞SW620、HT-29,人宫颈癌细胞Hela,人肺癌细胞A549和端粒酶阴性人胚肺成纤维细胞MRC-5由本室保存。②实验方法：利用定向克隆的方法分别构建由hTERT启动子驱动EGFP的质粒pLHCX-hTERT-EGFP和驱动wtp53表达的质粒pLHCX-hTERT-wtp53。以质粒pLHCX-hTERT-EGFP和课题组前期构建的质粒pLHCX-CMV-EGFP转染端粒酶阳性的人大肠癌细胞株SW620和HT-29、人宫颈癌细胞株Hela和人肺癌细胞株A549以及端粒酶阴性的正常人肺胚成纤维细胞MRC-5。③实验评估：通过流式细胞仪测定分析转染效率，证实pLHCX-hTERT-EGFP在不同细胞中的表达特异性。运用RT-PCR和Western Blot分别检测p53 mRNA和p53蛋白在p53突变的大肠癌细胞株SW620中表达。利用MTT检测质粒pLHCX-hTERT-wtp53对不同细胞的增殖抑制情况和流式细胞术检测不同细胞的凋亡情况。
结果:①通过酶切证实重组质粒pLHCX-hTERT-EGFP和pLHCX-hTERT-wtp53构建成功，并通过将pLHCX-hTERT-EGFP转染SW620细胞观察到绿色荧光蛋白的表达，测序证实构建质粒中包含完整的wtp53片段。②流式细胞术检测结果证实质粒pLHCX-hTERT-EGFP处理后端粒酶阳性的SW620、Hela和A549内增强型绿色荧光蛋白表达强度明显强于端粒酶阴性的
MRC-5（P < 0.05），而pLHCX-CMV-EGFP在端粒阳性和阴性的上述细胞中表达无明显差异（P > 0.05）。③RT-PCR和Western Blot分别证实了p53mRNA和p53蛋白表达。④MTT检测到重组质粒对端粒酶阳性的大肠癌细胞有明显的增殖抑制作用，而对端粒酶阴性的MRC-5细胞生长无明显影响（P < 0.05）。⑤流式细胞检测观察到重组质粒引起端粒酶阳性的大肠癌细胞凋亡要明显强于端粒酶阴性的MRC-5细胞（P < 0.05）。
结论:构建的反转录病毒载体质粒pLHCX-hTERT-EGFP在端粒酶阳性的肿瘤细胞中特异性表达；重组反转录病毒载体质粒pLHCX-hTERT-wtp53转染后对端粒酶阳性的大肠癌细胞有特异性的影响作用。
关键词:人端粒酶逆转录酶基因启动子；野生型p53；基因治疗

余新，洪葵，邓俊晖，刘天德，雷钧，张吉翔，邵江华.首次应用反转录病毒仅感染分裂细胞特性构建hTERT驱动野生型p53基因反转录病毒载体质粒[J].中国组织工程研究与临床康复，2008，12(3):493-497
[www.zglckf.com/zglckf/ejournal/upfiles/08-3/3k-493(ps).pdf]