hTRsiRNA腺病毒构建及其体外抗肿瘤初步探究.docVIP

hTRsiRNA腺病毒构建及其体外抗肿瘤初步探究 　 作者：李燕, 肖丽英, 姚刚, 李虹, 杨志伟, 李婉宜, 蒋忠华, 李明远 【摘要】 　　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siRNA, 表达靶向hTR的小干扰RNA分子（siRNA）, 用100 MOI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time PCR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。 【关键词】 hTR 腺病毒 RNA干扰 端粒酶 抗肿瘤作用 　　Construction of adenovirus expressing siRNA against hTR and its antitumor activity in vitro 　　LI Yan, XIAO Liying, YAO Gang, LI Hong, YANG Zhiwei, LI Wanyi, JIANG Zhonghua, LI Mingyuan* 　　State Key Laboratory of Oral Diseases, Sichuan University, Department of Microbiology, West China School of Preclinical and Forensic Medicine, Sichuan University, Chengdu 610041; Chengdu Institute of Biological Products, Chengdu 610063, China 　　[Abstract] AIM: To study the effect of the hTRsiRNA adenovirus on hTR mRNA gene silence, telomerase activity inhibition and antitumor in vitro. METHODS: RNAi adenovirus vector, AdhTRsiRNA, and negative control AdNTsiRNA were constructed by an improved ligation method. Different tumor cells and liver cell line, HL7702, were infected with 100 MOI of the recombinant adenoviruses. TRAPELISA, Realtime PCR and FCM were used to analyze telomerase activity, hTR mRNA, apoptosis rate and hTERT protein expression. RESULTS: As compared with AdNTsiRNA, AdhTRsiRNA reduced both hTR mRNA levels (70.21%) and telomerase activity (58.87%) of HeLa cells significantly, increased apoptosis rate (29.7%). But the telomerase activity of HL7702 and hTERT protein didn’t show the tendency of decrease. CONCLUSION: It is supposed that the hTRsiRNA adenovirus could knockdown hTR gene specifically and suppress the tumor cell growth in vitro efficiently. M