荷载hTERT基因double-shRNA溶瘤腺病毒对人前列腺癌DUl45细胞的杀伤效果

The antitumor effects of oncolytic adenovirus ZD55-double-hTERT expressing double small hairpin RNA targeting hTERT gene on prostate cancer DUl45 cells

目的 构建荷载hTERT基因double-shRNA溶瘤腺病毒ZD55-double-hTERT,研究其对前列腺癌DUl45细胞的杀伤效果.方法 实时荧光定量PCR法检测对hTERT基因的mRNA表达的影响;Western blot检测对前列腺癌细胞hTERT、病毒E1A蛋白表达的影响;MTT法检测对细胞增殖的抑制作用;Hoechst-33342染色了解不同处理组对前列腺癌细胞凋亡的诱导作用.结果 成功包装病毒ZD55-double-hTERT;RT-PCR和Western blot结果显示：ZD55-double-hTERT组hTERT表达量明显减少,与其他各组比较差异有统计学意义（P＜0.05）,所包装的病毒可以表达E1A蛋白.MTT结果显示：20MOI的各组病毒感染DUl45细胞72 h后,ZD55-double-hTERT组细胞存活率为（45.13±3.41）％,显著低于Blank组（81.60±3.31）％、ZD55-hTERT1组（70.51±4.69）％和ZD55-hTERT2组（76.93±1.63）％（P＜0.05）.Hoechst-33342染色结果显示：ZD55-double-hTERT（57.29±4.19）％与Blank（3.29±1.73）％、ZD55-hTERT1（23.14±3.56）％、ZD55-hTERT2（33.38±3.55）％相比,DU145细胞凋亡率明显增加（P＜0.05）.结论 荷载hTERT基因double-shRNA溶瘤腺病毒ZD55-double-hTERT与单靶点溶瘤腺病毒比较,对前列腺癌DUl45细胞hTERT基因的沉默作用增强,抑制增殖和促进凋亡的作用进一步提高.

Objective To investigate the antitumor effects of oncolytic adenovirus ZD55-double-hTERT expressing double small hairpin RNA targeting hTERT gene on prostate cancer DUl145 cells.Methods The inhibitory effect of ZD55-double-hTERT on hTERT expression was evaluated by quantitative real-time PCR and Western blot analysis.The expression of E1A protein in DUl45 cell infected with viruses respectively was detected by Western blot,The cytotoxicity of ZD55-double-hTERT was detected by MTT assay.The apoptosis effect was determined by Hoechst-33342.Results Real-time PCR and Western blot assay showed that ZD55-double-hTERT could significantly decrease the expression of hTERT in DUl45 cells.Western blot assay of the E1A expression indicated DUl45 cells infected with adenoviruses could express E1A,but blank group could not.MTT assay showed that cell viability of ZD55-double-hTERT group was significantly lower than that in other groups.ZD55-doublehTERT treatment of DU145 cells increased apoptotic cell death as compared to ZD55-hTERT1,ZD55-hTERT2 and blank group.Conclusions Oncolytic adenovirus ZD55-double-hTERT can more effectively knock down the expression hTERT in DUl45 cells,and exert more dramatically antitumor effects than ZD55-hTERT1 and ZD55-hTERT2.