抑制ATM表达对^(60)Co照射下宫颈癌细胞损伤修复机制的影响  

Influence of siRNA targeting against ATM on DNA damage repair for cervical carcinoma cells after ^(60)Co irradiation

作　　者：魏莉[1] 辛晓燕[1] 王建[1] 薛涛[2] 曹云新[3] 雷迎锋[4] 张红菊[1] 

机构地区：[1]第四军医大学西京医院妇产科,西安710032 [2]第四军医大学西京医院耳鼻喉科,西安710032 [3]第四军医大学西京医院基础部免疫学教研室,西安710032 [4]第四军医大学西京医院基础部微生物学教研室,西安710032

出　　处：《肿瘤》2007年第8期607-610,共4页Tumor

摘　　要：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 study the influence of RNA interference targeting against ataxia-telangiectasia mutated （ATM） gene on DNA damage repair for human cervical carcinoma HeLa cells after ^60Co irradiation. Methods： Recombinant eukaryotic expression plasmid pSup-ATM was transfected into human cervical carcinoma HeLa cells by electroporation. Stable cell line was screened by G418 selection. The inhibition of ATM was determined by semi-quantitative RT-PCR, Western blot, and immunofluorescence method. The expression of ATM, phospho-p53 （Ser15）, phospho-CHK2 （Thr68）, p53, and CHK2 protein were determined by Western blotting and cell cycle was monitored by flow cytometry （FCM） after ^60Co irradiation. Results： The expression level of ATM gene was significantly lower in the HeLa^ATM cells than that in HeLa and HeLa^ns cells. The cervical carcinoma model with low expression of ATM was successfully established. The expression levels of phospho-p53 （Serl5） , phospho-CHK2 （Thr68）, and p53 protein were all significantly decreased in the HeLaATM cells after ^60Co irradiation. FCM showed that the cells were accumulated in G2 phase and the proportion of cells in G1 phase was reduced. Conclusion： The RNA interference targeting against ATM gene significantly inhibites DNA repair response of cervical carcinoma HeLa^ATM cells after ^60Co irradiation. The mechanism is related with deletion of ATM protein expression and blockade of ATM-dependent DNA repair pathway.

关 键 词：宫颈肿瘤 放射疗法 RNA 小分子干扰 ATM基因 HELA细胞 

分 类 号：R737.33[医药卫生—肿瘤]