rPB-DR(6)调控的溶瘤腺病毒对前列腺癌细胞的杀伤作用  

Antitumor effect of rPB-DR(6) promoter-regulated oncolytic adenovirus on prostate cancer cells

作　　者：付成伟[1] 王洛夫[1] 江军[1] 孙中义[1] 兰卫华[1] 张克勤[1] 

机构地区：[1]第三军医大学大坪医院野战外科研究所泌尿外科,重庆400042

出　　处：《第三军医大学学报》2013年第24期2659-2664,共6页Journal of Third Military Medical University

基　　金：重庆市自然科学基金(CSTC2010BB5164)~~

摘　　要：目的构建含rPB-DR(6)启动子和CD自杀基因的重组溶瘤腺病毒,观测其对雄激素依赖性和非依赖性前列腺癌细胞的杀伤作用,探索雄激素非依赖性前列腺癌治疗的新方法。方法应用分子生物学技术,构建并包装出重组溶瘤腺病毒Ad5-rPC,纯化并扩增,测定滴度。采用PCR方法对重组溶瘤腺病毒颗粒进行鉴定并测序,Western blot检测E1A、CD蛋白表达,TCID50法检测重组腺病毒感染癌细胞后的复制能力,应用CCK-8法检测重组病毒特异性杀伤能力。结果经PCR及测序鉴定证实成功构建了包含rPB-DR(6)启动子和CD基因的重组溶瘤腺病毒Ad5-rPC,测滴度为1.1×1011pfu/mL。Western blot检测到E1A和CD蛋白阳性表达。Ad5-rPC病毒感染前列腺癌细胞系细胞产生的子代病毒滴度均大于3.0×106pfu/mL,CCK-8法证实重组腺病毒对雄激素依赖性和非依赖性前列腺癌细胞均具有特异性杀伤能力,杀伤能力均大于65%。结论前列腺特异性启动子调控的溶瘤腺病毒能在前列腺癌细胞内靶向复制,并对雄激素依赖性和非依赖性前列腺癌细胞均有特异性杀伤作用。Objective To construct conditionally replicating oncolytic adenovirus regulated by prostate specific promotor rPB-DR（6） with an insertion of cytosine deaminase （CD） gene driven by mCMV promoter, to observe its antitumor effect, and to explore a new method for the treatment of androgen-independent prostate cancer. Methods Recombinant oncolytic adenovirus Ad5-rPC was constructed and packaged using molecular biotechniques, identified by PCR and sequencing, purified by CSCI gradient centrifugation, and tested for PFU titer detection. Expression of E1A and CD proteins in the adenovirus-transfected prostate cancer cells was detec- ted by Western blotting. Antitumor effect of the recombinant adenovirus on prostate cancer cells was tested by CCK-8 assay. Results By PCR and restriction endonuclease assay, rPB-DR（6） promoter and CD gene driven by mCMV promoter were inserted into recombinant adenovirus successfully. The titer of the purified recombinant adenovirus Ad5-rPC was 1.1 × 10^11 pfu/mL. Western blot results showed positive expression of E1A and CD proteins in transfected prostate cancer cells. The titer of the progeny viruses produced by prostate cancer cells which were transfected by Ad5-rPC was greater than 3.0 × 106 pfu/mL. CCK-8 assay and cell counting method confirmed that Ad5-rPC had antitumor effects on both androgen-dependent and androgen-independent prostate cancer cells. Conclusion Recombinant oncolytic adenovirus regulated by prostate specific promoter has the ability of reproduction in prostate cancer cells uniquely, and can specifically kill prostate cancer cells.

关 键 词：溶瘤腺病毒 rPB—DR(6)启动子 CD基因 前列腺癌 基因治疗 

分 类 号：R394-33[医药卫生—医学遗传学] R73-362[医药卫生—基础医学]