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  

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

作　　者：李玮[1] 林洪义[1] 刘贤锡[2] 王伟[2] 

机构地区：[1]山东大学控制科学与工程学院生物医学工程研究所,济南250061 [2]山东大学医学院生化与分子生物学研究所,济南250012

出　　处：《山东大学学报（医学版）》2010年第6期36-40,共5页Journal of Shandong University:Health Sciences

基　　金：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)

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pAd-PL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。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 the pMD19-T simple vector followed by ligation with the PGL3-PSES fragment and then recombined with the 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.

关 键 词：鸟氨酸脱羧酶 S-甲硫氨酸脱羧酶 前列腺肿瘤 

分 类 号：R641[医药卫生—外科学]