论文部分内容阅读
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 effect and mechanism of RNA interference (RNAi) combined with radiation on nude mice xenografts. Methods: Small interfering RNA (siRNA) targeted to hTERT gene was designed and constructed. The recombinant plasmid hTERT-pGenesil was constructed and transfected into U251 cells. In combination with 2Gy γ-ray irradiation, TRAP-PCR-ELISA and comet assay were used to detect telomerase activity and DNA single-strand breaks simultaneously in control group, RNAi group, radiotherapy group and RNAi combined with radiotherapy group Tail moment). Results: There was no tail (P = 0.796) in control group and RNAi group, which indicated that RNAi itself did not cause single strand break (SSB); while both radiotherapy group and RNAi combined radiotherapy group showed obvious comet images Compared with radiotherapy group, the coma of RNAi combined radiotherapy group was significant (P <0.05). The telomerase activity (A value) of control group, RNAi treatment group, radiotherapy group and RNAi combined with radiotherapy group were 1.182 ± 0.019, 0.578 ± 0.034, 1.394 ± 0.027 and 0.871 ± 0.031 respectively, which showed that siRNA had a decreased telomerase activity (P <0.05), while the 2Gy radiotherapy increased the telomerase activity, P <0.05. Conclusion: Inhibition of human uterine glioma hTERT gene expression by RNA interference, reducing telomerase activity and reducing DNA damage repair, and thus have a significant radiosensitizing effect on human glioma in nude mice.