hTRsiRNA腺病毒的构建及其体外抗肿瘤的初步研究.doc

文档介绍：hTRsiRNA腺病毒的构建及其体外抗肿瘤的初步研究
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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, %, %, %, 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 binant adenoviruses. TRAPELISA, Realtime PCR and FCM were used to analyze telomerase activity, hTR mRNA, apoptosis rate and hTERT protein expression. RESULTS: pared with AdNTsiRNA, AdhTRsiRNA reduced both hTR mRNA levels (%) and telomerase activity (%) 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 e