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

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。

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

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.