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-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。
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. TRAPELISA, 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 teIomerase activity of HL-7702 and hTERT protein didnt 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. Maybe this siRNA expressing recombinant adenovirus system could be a new method for cancer gene therapy.
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2008年第6期570-573,共4页
Chinese Journal of Cellular and Molecular Immunology
基金
国家教育部博士点基金资助项目(20040610050)
