RNA干扰技术对裸鼠人胶质瘤放射增敏及其机制的探讨

Mechanism of radiosensitization of RNA interference in transplanted human glioma in nude mice

目的:探讨RNA干扰(RNAi)联合放射对裸鼠移植瘤的作用及其机制。方法:设计靶向hTERT基因的小干扰RNA(siRNA),构建重组质粒hTERT-pGenesil并转染U251细胞,将其种植于裸鼠从而建立裸鼠移植瘤模型。在RNA干扰时联合2Gyγ射线照射,采用TRAP-PCR-ELISA法和彗星电泳分析方法同时检测对照组、RNAi治疗组、放射治疗组及RNAi联合放射治疗组的端粒酶活性和DNA单链断裂(尾矩)。结果:对照组与RNAi治疗组细胞无彗尾(P=0.796),表明RNAi本身不会导致DNA单链断裂(SSB);而放射治疗组和RNAi联合放射治疗组均出现明显的彗星图像,且RNAi联合放射治疗组的彗尾较放射治疗组显著,P<0.05。对照组、RNAi治疗组、放射治疗组及RNAi联合放射治疗组的端粒酶活性(A值)分别1.182±0.019、0.578±0.034、1.394±0.027和0.871±0.031,显示siRNA有减少端粒酶活性的作用(P<0.05),而2Gy放疗使端粒酶活性有所升高,P<0.05。结论:通过RNA干扰技术抑制裸鼠人胶质瘤hTERT基因的表达,降低端粒酶活性,减少DNA损伤的修复,从而对裸鼠人胶质瘤有明显的放疗增敏作用。

OBJECTIVE： To investigate the effoct and mecha nism of RNA interference combined with ionizing radiation for truman glioma. METHODS： Small interference RNA （siRNA） targeting human telomerase reverse transcriptase （hTERT） gene was designed. Then hTERT-pGenesil vector was constructed and transfected into human glioma. U251 cells were transplanted into nude mice tn con struct human glioma model. RNAi combined 2 Gy y-irradiation was utilized to deaf with nude mice. The change of telomerase activity and DNA single-strand breaks in all control group, RNA interference group, radiation group and RNA interference combined with radia tion group were determined by PCR based telomeric repeal amplification protocol （TRAP） coupled with ELISA and Comet assay. RESULTS： Comet image was not observed in control group and RNAi group （P= 0. 796）. It showed that RNAi itself didn＇t resuh to DNA singlestrand breaks. While radiation group and RNA interference combined with radiation group all showed obvious comet image. The SSB of RNAi combined with radiation group compared with radiation group was more obvious （P〈0.05）. By examining the telomerase activity of human glio ma in nude mice, the value in control group, RNAi group, radiation group and RNAi combined with radiation group were 1. 182 ± 0. 019, 0. 578±0. 034, 1.394±0.027 and 0. 871± 0. 031 respectively, which suggested that siRNA could inhibit the activity of tumor telomerase （P〈 0.05）, and 2 Gy y-irradiation could increase the activity of tumor telomerase （P〈0. 05）. CONCLUSION： By RNA interference the hTERT gene expression was inhibited, the telomerase activity was decreased, the of DNA breaks were repaired, and human glioma was sensitized to ionizing radiation in nude mice.