【摘要】 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。更多还原

【Abstract】 OBJECTIVE:To investigate the effect and mechanism of RNA interference combined with ionizing radiation for human 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 to construct human glioma model.RNAi combined 2 Gy γ-irradiation was utilized to deal 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 radiation group were determined by PCR-based telomeric repeat 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 result to DNA single-strand 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 glioma 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 γ-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.更多还原