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目的:构建RNA干扰(RNAi)腺病毒,通过体外实验观察其抑制肿瘤细胞中人端粒酶RNA(hTR)基因表达、降低瘤细胞中的端粒酶活性、以及促进肿瘤细胞凋亡等作用。方法:首先用一种新的体外连接法构建腺病毒Ad-hTR-siR-NA,表达靶向hTR的小干扰RNA分子(siRNA),用100MOI的重组腺病毒感染不同的肿瘤细胞系,再用TRAP-ELISA法测定细胞端粒酶活性、Real-timePCR测定hTR的mRNA水平、流式细胞术(FCM)测定肿瘤细胞凋亡率及人端粒酶反转录酶(hTERT)的蛋白变化。结果:感染了Ad-hTR-siRNA的肿瘤细胞的端粒酶活性明显降低,其中以HeLa细胞降低最明显,其端粒酶活性抑制率达到58.87%,hTR的mRNA表达下调70.21%,细胞凋亡率为29.7%,但hTERT的蛋白表达并不被阻断。结论:Ad-hTR-siRNA可以有效、特异地抑制hTR基因表达,诱导体外培养的肿瘤细胞凋亡,具有抗肿瘤活性;此结果提示Ad-hTR-siRNA在肿瘤基因治疗方面具有潜在的应用价值。
OBJECTIVE: To construct RNA interference (RNAi) adenovirus, and to observe the effect of inhibiting telomerase RNA (hTR) gene expression in tumor cells, reducing the telomerase activity in tumor cells and promoting the apoptosis of tumor cells in vitro. Methods: A novel adenovirus Ad-hTR-siR-NA was constructed and a small interfering RNA (siRNA) targeting hTR was expressed. A new adenovirus containing 100 MOI was used to infect different tumor cell lines. The telomerase activity was measured by TRAP-ELISA, the mRNA level of hTR was determined by Real-time PCR, the apoptosis rate of tumor cells and the protein expression of human telomerase reverse transcriptase (hTERT) by flow cytometry (FCM). Results: The telomerase activity of tumor cells infected with Ad-hTR-siRNA was significantly decreased, the most significant decrease was HeLa cells, the inhibition rate of telomerase activity was 58.87%, the mRNA expression of hTR was down-regulated 70.21%, apoptosis The rate was 29.7%, but hTERT protein expression was not blocked. Conclusion: Ad-hTR-siRNA can effectively and specifically inhibit the expression of hTR gene and induce the apoptosis of tumor cells cultured in vitro with antitumor activity. The results suggest that Ad-hTR-siRNA has potential value in the gene therapy of cancer.