DNA损伤修复基因与人鼻咽癌细胞辐射耐受CNE-2R细胞的相关性研究  

Relationship between DNA damage repair and radioresistance in nasopharyngeal carcinoma CNE-2 Rcells

作　　者：王亚利[1] 马秀龙[1] 任宏涛[1] 王宝娜[1] 王中卫[1] 

机构地区：[1]西安交通大学医学部第二附属医院肿瘤科,陕西西安710004

出　　处：《西安交通大学学报（医学版）》2015年第2期174-179,共6页Journal of Xi’an Jiaotong University（Medical Sciences）

基　　金：陕西省科技发展计划项目(No.2012K13-01-09)~~

摘　　要：目的探讨射线诱导后DNA损伤修复相关基因在放射敏感性不同鼻咽癌细胞系CNE-2、CNE-2R的差异表达。方法中性彗星分析法检测细胞DNA的双链断链(DSB);免疫荧光技术检测磷酸化组蛋白γH2AX焦点形成、射线照射后细胞放射性损伤的时间剂量效应及放射敏感性的变化;基因芯片(OHS-029)技术检测CNE-2及CNE-2R细胞2Gy照射前后的基因差异表达;Western blot方法验证二者的差异表达蛋白质。结果 4Gy的9MeV-β射线照射后2h,CNE-2R细胞与CNE-2细胞相比,细胞DNA损伤程度加重,且随时间的延长更为明显。荧光显微镜显示,2Gy的9MeV-β射线照射后6h,CNE-2各时间段细胞γH2AX阳性细胞率明显高于CNE-2R组。DNA损伤相关基因表达谱分析发现,CNE-2和CNE-2R细胞相比,差异表达的DNA损伤修复相关基因共37个,其中上调基因24个,下调13个,其中6个位点差异6倍以上,3个位点低于0.1的差异表达;Western blot结果显示,与CNE-2细胞相比,GADD45a、RRAD1在CNE-2R中的表达水平明显下调,而ERCC1及PRKDC蛋白质明显上调,与基因芯片的研究结果一致。结论 9MeV-β射线照射后CNE-2细胞DNA的损伤程度较CNE-2R细胞明显,CNE-2R细胞放射抗拒性与DNA损伤修复能力的基因差异表达相关。通过对筛选出的靶基因进行调控,有可能改变细胞的放射敏感性。Objective To investigate the differential gene expression of nasopharyngeal carcinoma cell line CNE-2 and CNE-2R exposed to X-rays.Methods We used neutral comet assay to detect the intrinsic radiosensitiv-ity of tumor cells,immunofluorescence to detect the phosphorylated histone γH2AX and Rad5 1 foci formation and to assay the time-dose effects on cell radiation injury and radiation radiosensitivity change after irradiation.The dif-ferential gene expression was screened with cDNA microarray by DNA damage repair related gene chip.Differenti-ally expressed proteins screened were confirmed by Western blot.Results After 2 h of 4 Gy 9MeV-β-ray irradia-tion,CNE-2R cells had aggravated DNA damage compared to CNE-2 cells and the damage was more obvious with the prolongation of time.It suggested that cell DNA damage severity was more obvious.The immunofluorescence showed the positive rates ofγH2AX cells in CNE-2 cell line were higher than those in CNE-2R cell line.There were 37 different genes related to DNA damage repair in CNE-2 cell line compared with CNE-2R cell line,while 24 up-regulated and 13 down-regulated genes were found in SiHaR cells.In them,there were 6 genes whose ratios were 6 times higher and 3 genes whose ratios were lower than 0.1.Western blot verified the results of four differentially expressed proteins compared with CNE-2 cells,in which GADD45a and RRAD1 expression levels were significantly lowered in CNE-2R while the RAD9a and RCF2 protein significantly increased.Conclusion The radioresistance of CNE-2R cells is significantly related to DNA damage repair capacity in certain mutations at the gene level.It is possible to regulate cell radiosensitivity by regulating the related gene.

关 键 词：鼻咽癌 DNA 损伤修复 基因表达谱 放射抗拒性 

分 类 号：R739.6[医药卫生—肿瘤]