前列腺非雄激素依赖启动子介导的双反义腺病毒的制备及组织特异性检测

山东大学学报(医学版) ›› 2010, Vol. 48 ›› Issue (6): 36-40.

前列腺非雄激素依赖启动子介导的双反义腺病毒的制备及组织特异性检测

李玮1, 林洪义1,刘贤锡2,王伟2

山东大学 1. 控制科学与工程学院生物医学工程研究所, 济南 250061;
2. 医学院生化与分子生物学研究所, 济南 250012

收稿日期:2010-02-03 出版日期:2010-06-16 发布日期:2010-06-16
通讯作者: 刘贤锡，博士生导师，主要从事分子生物学研究。 Email: xianxi@sdu.edu.cn
作者简介:李玮(19- )，女，博士研究生，讲师，主要从事分子生物学研究。
基金资助:
国家自然科学基金资助项目（30900730）；山东省自然科学基金资助项目（Q2007D01）；山东省优秀中青年科学家基金资助项目（2006BS01011）。

Construction and identification of a prostatic androgen-independent promotermediated ODC and AdoMetDC antisense RNA-expressing adenovirus

LI Wei1，LIN Hongyi1，LIU Xianxi2，WANG Wei2

1. Biomedical Engineering Institution, School of Control Science and Engineering, Shandong University, Jinan 250061, China；
2. Institute of Biochemistry and Molecular Biology，School of Medicine，Shandong University, Jinan 250012, China

Received:2010-02-03 Online:2010-06-16 Published:2010-06-16

摘要/Abstract

摘要：

目的制备前列腺非雄激素依赖启动子（PSES）介导的鸟氨酸脱羧酶(ODC)和S-腺苷甲硫氨酸脱羧酶(AdoMetDC)双反义腺病毒。方法构建pShuttle-Basic-PSES AdoMetDC-ODC-PolyA穿梭载体，并与载体pAdPL进行体外重组，包装成重组腺病毒。将重组腺病毒转染至前列腺癌、直肠癌等癌细胞中，噻唑兰比色（MTT）法观察其对细胞增殖的影响，Western Blot检测重组腺病毒对细胞中ODC和AdoMetDC表达的抑制作用。结果成功构建了前列腺特异的ODC和AdoMetDC双反义RNA表达载体，并包装制备出能特异的在前列腺细胞中表达产生ODC和AdoMetDC反义RNA的重组腺病毒。该重组腺病毒具有前列腺组织特异性。结论构建的前列腺非雄激素依赖启动子介导的ODC、AdoMetDC双反义腺病毒具有组织特异性。

关键词: 鸟氨酸脱羧酶;S甲硫氨酸脱羧酶;前列腺肿瘤

Abstract:

Objective To construct a recombinant adenovirus that can simultaneously express antisense ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC) specifically in prostate cancer cells and to evaluate its tissue-specific expression. Methods Fragments of ODC and AdoMetDC cDNAs were inserted into pMD19-T simple vector followed by ligation with PGL3- PSES fragment and then recombined with pShuttle-Basic vectors in ADxsi cells. The AdPSES-ODC-AdoMetDCas virus was produced in HEK293 cells. Western blot，cancer cell invasion and MTT analysis were applied to detect ODC and AdoMetDC expressions in tumor cells. Results The recombinant adenovirus, packaged and amplified in HEK293 cells， was successfully constructed. Western blot，cancer cell invasion and MTT analysis results showed that expressions of ODC and AdoMetDC were significantly lower in prostate cancer cells infected with the recombinant adenovirus than in HT29 cells infected with the same virus. Conclusion The recombinant adenovirus can specifically deplete ODC and AdoMetDC expressions in prostate cells and the decreased expressions have tissuespecificity.

Key words: Ornithine decarboxylase; Sadenosylmethionine； Prostate neoplasms

中图分类号:

R641

李玮1, 林洪义1,刘贤锡2,王伟2. 前列腺非雄激素依赖启动子介导的双反义腺病毒的制备及组织特异性检测[J]. 山东大学学报(医学版), 2010, 48(6): 36-40.

LI Wei1，LIN Hongyi1，LIU Xianxi2，WANG Wei2. Construction and identification of a prostatic androgen-independent promotermediated ODC and AdoMetDC antisense RNA-expressing adenovirus[J]. JOURNAL OF SHANDONG UNIVERSITY (HEALTH SCIENCES), 2010, 48(6): 36-40.

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