PTEN增强溶瘤腺病毒对前列腺癌细胞的特异性杀伤作用

摘要

目的 研究由survivin启动子调控、携带抑癌基因PTEN的条件复制型重组腺病毒对前列腺癌细胞的杀伤作用.方法 构建含有survivin启动子和PTEN基因的重组腺病毒reADGL3BSurvPTEN,感染前列腺癌细胞系和正常前列腺上皮细胞系.利用Western blot检测病毒感染前后前列腺癌细胞中PTEN的表达变化,CCK-8法及流式细胞学检测重组腺病毒对前列腺癌细胞生长及凋亡的影响.结果 PCR结果证实成功构建了含有survivin启动子和PTEN基因的腺病毒载体reADGL3BSurvPTEN,Western blot 结果显示感染ADGL3BSurvPTEN后在高表达survivin的前列腺癌细胞中PTEN表达明显增高,而在不表达survivin的正常前列腺细胞中,仅检测到内源性PTEN的表达.CCK-8法及流式细胞术结果表明reADGL3BSurvPTEN病毒对前列腺癌细胞的杀伤作用较不含PTEN基因的腺病毒reADGL3Bsurvivin更加明显,对正常前列腺上皮细胞没有显著的杀伤作用.结论 成功构建了含有survivin启动子和PTEN基因的条件复制型腺病毒,该病毒对前列腺癌细胞有显著的杀伤作用,实验结果为前列腺癌细胞靶向治疗提供了更为良好的条件复制型病毒载体及新的治疗策略.%Objective To construct a survivin-condittonally replicative oncolytic adenovirus expressing PTEN gene,and to evaluate its oncolytic effect on a human prostate cancer cell line LNCaP in vitro. Methods reADGL3BSurvPTFN containing survivin promotor and PTFN gene was constructed,and infected into LNCaP cells. The expression of PTFN in LNCaP cells was detected by Western blot, and growth inhibition, oncolytic effect and apoptosis of LNCaP cells were examined by CCK-8 and Annexiny/PI flow cytometry. Results The recombinant survivin-conditionally replicative oncolytic adenovirus expressing PTEN gene reADGL3BSurvPTEN was constructed successfully, which was confirmed by PCR. Western blot showed that PTFN null/survivin high prostate cancer cells infected with reADGL3BSurvPTFN expressed high level of PTFN , and survivin negative prostate epithelia cells infected with reADGL3BSurvPTEN only expressed endogenous level of PTEN. CCK-8 and flow cytometry revealed stronger oncolytic effect on LNCaP cells than reADGL3Bsurvivin which did not contain PTFN gene. reADGL3BSurvPTEN had no significant effect on survivin-negative normal prostate epithelia cells. Conclusion The successful construction of PTEN enhanced survivin-conditionally replicative oncolytic adenovtrus provides us a new strategy for prostate target therapy.