摘要
目的探讨不同基因型胶质母细胞瘤细胞系辐射抵抗性的差异及其原因。方法选取三种不同基因型GBM细胞系(U87MG、U251MG、U118MG),利用外显子测序技术检测P53、PTEN基因突变情况,蛋白免疫印迹检测MGMT表达情况。不同剂量(2、5、10 Gy)辐射后,采用流式细胞术检测细胞凋亡水平、平板克隆形成检测细胞增殖能力,分析其辐射抵抗性。利用蛋白免疫印迹检测DNA损伤标记蛋白(γH2AX)、NF⁃κB效应分子(RELA/p65)及EGFR在辐射后48 h内激活情况,分析DNA损伤与NF⁃κB及EGFR信号通路激活的相关性。结果U87MG、U251MG、U118MG均存在P53与PTEN的突变位点,其中U87MG、U251MG低表达MGMT,U118MG高表达MGMT。经过2、5、10 Gy辐射后48 h,各剂量均存在凋亡率变化值:U87MG>U118MG>U251MG,存活分数:U251MG>U118MG>U87MG。三种细胞系经辐射后γH2AX、RELA/p65、EGFR的激活均存在时间与剂量差异性。结论辐射抵抗性:U251MG>U118MG>U87MG,提示GBM细胞系辐射抵抗性与其基因型有关。三种细胞系辐射后NF⁃κB通路与EGFR通路的激活存在差异,最终导致其辐射抵抗性差异。
Objective To investigate the differences and causes of glioblastoma cell linesof different geno⁃typesin radiation resistance.Methods Three GBM cell lines with different genotypes(U87MG,U251MG,U118MG)were selected.P53 and PTEN gene mutations were detected by exon sequencing technology,and MGMT expression was detected by Western blotting.After irradiation withdifferent doses(2 Gy,5 Gy,10 Gy),flow cytometry was used to detect the levels of apoptosis,and plate clone formation to detect cell proliferations,so that the radia⁃tion resistance was analyzed.Western blot was used to detect the activation of DNA damage marker protein(γH2AX),NF⁃κB effector molecule(RELA/p65)and EGFR within 48 hours after radiation,followed byanalyz⁃ing the correlation between DNA damage and the activation of NF⁃κB and EGFR signaling pathways.Results U87MG,U251MG,and U118MG all hadmutation sites of P53 and PTEN.Among them,U87MG and U251MG low⁃expressed MGMT,and U118MG high⁃expressed MGMT.After 48 hradiation with the doses of 2 Gy,5 Gy,and 10 Gy,there was a change inthe apoptosis rate:U87MG>U118MG>U251MG,and at the survival score:U251MG>U118MG>U87MG across the four doses.There were differences in the activation time and dose ofγH2AX,RELA/p65 and EGFR in the three cell lines after irradiation.Conclusion Radiation resistance:U251MG has the highest radiation resistance,followed by U118MG and U87MG in sequence,which suggests that the radia⁃tion resistance of GBM cell line is related to its genotypes.There are differences in the activation of the NF⁃κB pathway and the EGFR pathway after irradiation to the three cell lines,which ultimately leads to differences in their radiation resistance.
作者
简海锋
梁惠平
岑柏宏
袁亚维
JIAN Haifeng;LIANG Huip-ing;CEN Bohong;YUAN Yawei(Department of Radiation Oncology,Affiliated Cancer Hospital of Guangzhou Medi-cal University,Guangzhou 510095,China)
出处
《实用医学杂志》
CAS
北大核心
2021年第12期1520-1528,共9页
The Journal of Practical Medicine
基金
国家自然科学基金青年项目(编号:82003210)
广东省基础与应用基础研究基金联合基金青年项目(编号:2020A1515110183)。
作者简介
通信作者:袁亚维,E⁃mail:yuanyawei@gzhmu.edu.cn。
