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目的:探讨抑制毛细血管扩张-共济失调突变基因(ataxia-telangiectasia mutated,ATM)表达对人宫颈癌细胞株HeLa在60Co照射下细胞损伤修复机制的影响。方法:电穿孔法将人ATM基因siRNA的重组真核表达质粒pSup-ATM转染宫颈癌HeLa细胞;G418筛选建立稳定转染株;RT-PCR、W estern-b lot、免疫荧光法检测ATM基因表达的抑制情况;W estern-b lot法检测60Co照射前后各组细胞ATM、p53Ser15磷酸化、CHK2Thr 68磷酸化、p53、CHK2蛋白的表达水平;流式细胞术检测60Co照射后各组的细胞周期变化。结果:ATM基因在HeLaATM细胞中表达明显低于在HeLa、HeLans细胞中的表达水平,成功地建立了ATM低表达的宫颈癌细胞模型。HeLaATM细胞在60Co照射后p53Ser15磷酸化、CHK2Thr68磷酸化、p53蛋白表达均显著降低,细胞周期呈现为G2期延长,G1/G2倒置。结论:抑制ATM基因表达可显著抑制宫颈癌细胞对60Co辐射损伤的修复。这一机制可能与HeLaATM细胞中ATM蛋白表达缺失,ATM依赖性的细胞损伤修复通路受阻有关。
OBJECTIVE: To investigate the effect of suppressing the expression of ataxia-telangiectasia mutated (ATM) on the cell repair mechanism of human cervical carcinoma cell line HeLa under 60Co irradiation. METHODS: The recombinant eukaryotic expression plasmid pSup-ATM of human ATM gene siRNA was transfected into HeLa cells by electroporation. Stable transfectants were screened by G418. RT-PCR and Western-blot were used to detect ATM gene The expression of ATM, p53Ser15 phosphorylation, CHK2Thr 68 phosphorylation, p53 and CHK2 protein in 60Co groups were detected by Western-blot before and after 60Co irradiation. Flow cytometry was used to detect the cell cycle Variety. Results: The expression of ATM gene in HeLaATM cells was significantly lower than that in HeLa and HeLans cells, and successfully established a low expression of ATM in cervical cancer cell model. The phosphorylation of p53Ser15, the phosphorylation of CHK2Thr68, the expression of p53 protein in HeLaATM cells after 60Co irradiation were significantly decreased, and the cell cycle showed prolongation of G2 phase and G1 / G2 upside down. Conclusion: Inhibition of ATM gene expression can significantly inhibit the cervical cancer cells on 60Co radiation injury repair. This mechanism may be related to the lack of ATM protein expression in HeLaATM cells and the blockage of ATM-dependent cell injury repair pathway.