【摘要】 目的:构建携带有Survivin启动子和报告基因的条件复制型腺病毒并观察该病毒对前列腺癌细胞的特异性溶瘤作用。方法:以前列腺癌细胞系LNCaP细胞全基因组DAN为模板.PCR扩增Survivin启动了,构建pGL3BSurvivin质粒表达载体,荧光素酶检测系统观察查列腺癌细胞中Survivin启动子活性。将pGL3BSurvivin亚克隆至穿梭质粒pShuule中,构建含有Survivin启动子的重组腺病毒reADGL3BSurvivin。转染HEK293细胞进行重组病毒的扩增、纯化和滴度检测。利用CCK- 8法检测重组腺病毒对前列腺癌细胞的生长抑制作用,以正常前列腺细胞为对照。结果:经多种限制性内切酶酶切、PCR及测序鉴定,证实成功构建了含Survivin启动子报告基因质粒表达载体。荧光素酶报告基因检测结果表明前列腺癌细胞内Survivin启动子活性明显高于正常细胞组。同时也证实成功构建了含Survivin启动子和报告基因的腺病毒载体;CCK-8结果显示reADGL3BSurvivin可有效抑制前列腺癌细胞增殖而对正常细胞无增殖抑制作用。结论:成功构建含Survivin启动子的条件复制型腺病毒具有选择性杀伤前列腺癌细胞的能力,实验结果为前列腺癌靶向治疗提供了良好的条件复制型病毒载体及新的治疗策略。更多还原

【Abstract】 Objective:To construct a conditional replicative oncolytic adenovirus regulated by survivin promotor.And to test its oncolytic effects on prostate cancer cell.Methods:Survivin promotor was amplified by PCR from the genome of-survivin positive prostate cancer cell line LNCaP.pGL3Bsurvivin plasmid was constructed and transfected into LNCaP cell,the survivin transcriptional responses was tested by Luciferase Reporter Gene Assay.pGL3Bsurvivin was subclone into pShuttle vector,and reADGL3Bsurvivin was constructed by homologous recombination with El null pAdEasy-1 in BJ5183.Adenoviral plasmid was propagated in293cells and purified.Oncolytic effect of reADGL3Bsurvivin on LNCaP was tested by CCK-8 test and flow cytometry.Results:Survivinconditional replicative oncolytic adenovirus reADGL3Bsurvivin was successfully constructed,which confirmed by PCR and luciferase reporter gene assay.CCK-8 test showed that reADGL3Bsurvivin could specifically inhibit proliferation and killed survivin positive prostate cancer cell line LNCaP cell, but showed no significant oncolytic effect on survivin negative normal prostate epithelia cell.Conclusions:Constructed reADGL3Bsurvivin could specifically killed survivin positive prostate cancer cell,which may he used in gene therapy against prostate cancer in the future.更多还原