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目的 观察表达人端粒酶逆转录酶小干扰RNA(hTERT-siRNA)的增殖腺病毒(ZD-hTERT)对人肝癌Bel-7402细胞增殖及凋亡影响.方法 ZD-hTERT、增殖腺病毒ZD-EGFP、表达hTERT-siRNA的增殖缺陷腺病毒Ad-hTERT、增殖缺陷腺病毒Ad-EGFP分别感染人肝癌Bel-7402细胞.Western blot法检测ElA表达;逆转录-聚合酶链反应(RT-PCR)、Western blot法检测hTERT表达;噻唑蓝(MTT)比色法检测细胞存活;结晶紫染色法检测细胞毒作用;原位末端标记法(TUNEL)检测凋亡.结果 感染ZD-hTERT、ZD-EGFP的Bel-7402细胞表达ElA;抑制hTERT表达作用依次为ZD-hTERT>Ad-hTERT>ZD-EGFP>Ad-EGFP;抑制Bel-7402细胞生长及细胞毒作用依次为ZD-hTERT>ZD-EGFP=Ad-hTERT>Ad-EGFP.感染ZD-hTERT、ZD-EGFP、Ad-hTERT、Ad-EGFP的Bel-7402细胞凋亡率(%)分别为(88.1±2.2)、(39.2±2.1)、(42.1±5.1)、(7.5±2.1),ZD-hTERT诱导凋亡作用最高(P<0.01).结论 表达hTERT-siRNA的增殖腺病毒能显著抑制人肝癌Bel-7402细胞hTERT基因表达,进而抑制其增殖,促进其凋亡.

Objective To evaluate the effects of suppressing hTERT gene expression on the cell proliferation and apoptosis of human hepatocellular carcinoma Bel-7402 cells with ZD-hTERT, an E1B-55KD gene-deleted conditionally replicative adenovirus armed with small interference RNA targeting hTERT gene. Methods Hepatocellular carcinoma Bel-7402 cells were infected with ZD-hTERT, ZD55-EGFP,an El B-55kd gene-deleted conditionally replicative adenovirus, Ad-hTERT, a replication-deficient adenovirus espressing hTERT-siRNA and Ad-EGFP, a replication-deficient adenovirus, respectively. El A protein expression was determined by Western blot. The hTERT expression levels of Bel-7402 cells were detected by RT-PCR and Western blot analysis respectively. Cell prohferation was assayed by MTr meth-ed. Bel-7402 cells were stained with crystal violet to assay tumor-selective eytotoxieity. Cell apoptosis was measured by TUNEL assay. Results Western blot assay of ElA expression indicated Bel-7402 cells in-fected with ZD-hTERT and ZD-EGFP could express ElA, but the cells infected with Ad-hTERT and Ad-EGFP could not. Significant reductions of hTERT mRNA and protein content were observed in lysates from Bel-7402 ceils infected with ZD-hTERT. Crystal violet stain and MTT assay demonstrated that the antitu-mot effect of ZD-hTERT was more potent than both ZD-EGFP and Ad-hTERT. ZD-hTERT treatment of Bel-7402 cells resulted in an increase of apoptotie cells as compared with ZD-EGFP and Ad-hTERT treat-ment. Conclusion ZD-hTERT can replicate selectively in Bel-7402 cells and result in cancer-specific cy-totoxicity. Conditionally replicative adenovirus armed with aiRNA against hTERT gene may prove to be a useful novel tool for renal cancer therapy.