Department of Urology, The Third Xiangya Hospital of Central South University, Department of Urology, The Third Xiangya Hospital, Yue-lu District, Changsha 410013, Hunan, China.
Chin J Cancer Res. 2013 Oct;25(5):593-9. doi: 10.3978/j.issn.1000-9604.2013.10.11.
To investigate the effects of adenovirus-mediated inducible nitric oxide synthase gene transfection on bladder transitional cell carcinoma T24 cells, and to provide novel insights and approaches to clinical therapies against bladder transitional cell carcinoma.
Firstly, construct recombinant adenovirus vector pAd-iNOS of iNOS, followed by transfection of pAd-iNOS into HECK293 packaging cells. Thirdly, harvest recombinant adenovirus rAd-iNOS after amplification and purification procedures. Finally, transfect the recombinant adenovirus rAd-iNOS into human bladder carcinoma T24 cells and examine the effect of rAd-iNOS transfection on apoptosis of T24 and possible mechanism.
As shown by this study, the recombinant adenovirus rAd-iNOS was constructed successfully. The virus titer was 5.8×10(8) PFU/mL and recombinant was verified by PCR analysis. Transfection of adenovirus rAd-iNOS into T24 cells could induce secretion of high NO concentration, P53 protein expression up-regulation, as well as promotion of T24 cell apoptosis.
The transfection of human bladder carcinoma T24 cells from recombinant adenovirus rAd-iNOS was confirmed to induce intracellular iNOS over-expression, high production of NO, up-regulation of intracellular P53 expression and promotion of cell apoptosis.
探讨腺病毒介导的诱导型一氧化氮合酶基因转染对膀胱移行细胞癌细胞的影响，为临床治疗膀胱移行细胞癌提供新的思路和方法。
首先构建 iNOS 的重组腺病毒载体 pAd-iNOS，然后将 pAd-iNOS 转染至 HECK293 包装细胞。第三步，经过扩增和纯化程序后收获重组腺病毒 rAd-iNOS。最后，将重组腺病毒 rAd-iNOS 转染入人膀胱癌细胞 T24 中，并检测 rAd-iNOS 转染对 T24 细胞凋亡的影响及可能的机制。
本研究成功构建了重组腺病毒 rAd-iNOS。病毒滴度为 5.8×10(8)PFU/mL，经 PCR 分析证实重组。腺病毒 rAd-iNOS 转染 T24 细胞可诱导高浓度 NO 的分泌、P53 蛋白表达上调以及促进 T24 细胞凋亡。
证实重组腺病毒 rAd-iNOS 转染人膀胱癌细胞可诱导细胞内 iNOS 过表达、NO 大量产生、细胞内 P53 表达上调并促进细胞凋亡。
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