携带白细胞介素24基因溶瘤腺病毒对膀胱癌Biu87细胞的杀伤效应

目的 观察携带人白细胞介素(IL)24基因的溶瘤腺病毒(ZD55-IL24)对膀胱癌Biu87细胞的杀伤效应.方法 聚合酶链反应(PCR)鉴定溶瘤腺病毒ZD55-IL24并扩增、纯化和滴度测定.ZD55-IL24感染人膀胱癌Biu87细胞系,Western blot法检测腺病毒E1A和IL24蛋白的表达情况;原位末端标记法(TUNEL)检测细胞凋亡;通过结晶紫染色法检测细胞杀伤作用;噻唑蓝(MTT)法检测细胞存活情况.结果 PCR鉴定说明ZD55-IL24包含目的 基因且无野生型腺病毒污染;Western blot结果表明ZD55-IL24能在肿瘤细胞内表达E1A并高效介导IL24基因在Biu87细胞中的表达;TUNEL法显示ZD55-IL24能显著诱导Biu87细胞的凋亡,ZD55-IL24、ZD55-EGFP、Ad-IL24处理的Biu87细胞凋亡率分别为(52.3±3.2)%、(26.3±2.3)%、(32.0±3.1)%.结晶紫染色结果表明ZD55-IL24对Biu87细胞有明显的杀伤作用,MTT表明用MOI=10的ZD55-IL24、ZD55-EGFP和Ad-IL24感染Biu87细胞,4 d后存活率分别为(27.6±1.3)%、(48.7±2.5)%和(59.7±3.53)%.结论 成功获得纯化的溶瘤腺病毒ZD55-IL24,并进一步证明ZD55-IL24可在膀胱癌细胞内选择性地增殖并诱导膀胱癌Biu87细胞的凋亡,显示出良好的抗肿瘤效果.

Antitumor effects of oncolytic adenovirus expressing IL24 on bladder cancer Biu87 cells in vitro

Objective To study the antitumor effects of oncelytic adenovirus expressing human IL24 gene (ZD55-IL24) on human bladder cancer Biu87 ceils. Methods The recombinant adenoviruses ZD55-IL24 were verified by PCR. Further viruses were propagated, purified and functional PFU titens were determined by plaque assay on 293 cells. The expression of E1A and IL24 was detected by Western blot a-nalysis. The apoptosis was determined by TdT mediated dUTP nick end labeling (TUNEL). ZD55-IL24 were used to infect Biu87 cells. The inhibition of Biu87 cell proliferation by ZD55-IL24 was determined by MTI" assay and crystal violet dye method at different MOI values. Results The analysis of PCR indicated the recombinant adenovirus ZD55-IL24 containing IL24 gene without wild adenovirus. Functional PFU ti-tens of ZD55-IL24 were 2.0 × 1010 PFU/ml. Western blot analysis confirmed that ZD55-IL24 could express IL24 and E1A protein in Biu87 cell line with high efficiency. The percentage of the apoptotic cells was (52.3±3.2) %, (26.3±2.3) % and (32.0±3.1) % respectively after treatment with ZD55-IL24,ZD55-EGFP and Ad-IL24 for 48 h. Crystal violet staining and MTT assay showed ZD55-IL24 could induce obviously cytotoxic effects and growth inhibition of Biu87 cells. Cell viability rate was (27.6±1.3) %, (48.7±2.5) % and (59.7±3.53) %, respectively after tumor cells were infected with ZD55-IL24, ZD55-EGFP and Ad-IL24 at MOI = 10 on the day 4 postinfection. Conclusion The recombinant adenovi-rus ZD55-IL24 was successfully obtained. ZD55-IL24 could exert potential antitumor effects and offer a no-vel approach for gene therapy of bladder cancer.