摘要
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E1A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E1A；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 ceils 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 E1B-55kd gene-deleted conditionally replicative adenovirus, Ad-hTERT, a replication-deficient adenovirus espressing hTERT-siRNA and Ad-EGFP, a replication-deficient adenovirus, respectively. E1A 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 proliferation was assayed by MTT method. Bel-7402 cells were stained with crystal violet to assay tumor-selective cytotoxicity. Cell apoptosis was measured by TUNEL assay. Results Western blot assay of E1A expression indicated Bel-7402 cells infected with ZD-hTERT and ZD-EGFP could express E1A, but the cells infected with Ad-hTERT and AdEGFP could not. Significant reductions of hTERT mRNA and protein content were observed in lysates from Bel-7402 cells infected with ZD-hTERT. Crystal violet stain and MTT assay demonstrated that the antitumot 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 apoptotic cells as compared with ZD-EGFP and Ad-hTERT treatment. Conclusion ZD-hTERT can replicate selectively in Bel-7402 cells and result in cancer-specific cytotoxicity. Conditionally replicative adenovirus armed with siRNA against hTERT gene may prove to be a useful novel tool for renal cancer therapy.
出处
《中华实验外科杂志》
CAS
CSCD
北大核心
2008年第11期1385-1387,共3页
Chinese Journal of Experimental Surgery
基金
国家自然科学基金资助项目（30772174）
江苏省自然基金资助项目（BK2006036）
江苏省高校自然基金资助项目（06KJB320114）
