肺癌放射抗性细胞系构建及生物行为研究  

Establishment and biological characterization of radiation-resistant lung carcinoma cell lines

作　　者：邱祥南 张伟[1,2] 李浩[1] 覃朝晖[3] 王侠[1] 章龙珍[1] 姚元虎[1] 

机构地区：[1]徐州医学院附属医院放射治疗科,221002 [2]徐州医学院肿瘤生物治疗实验室 [3]徐州医学院公共卫生学院

出　　处：《中华放射肿瘤学杂志》2015年第6期703-707,共5页Chinese Journal of Radiation Oncology

基　　金：江苏省“六大人才高峰”项目（2013WSN082）;江苏省卫生厅科技项目（H201426）;徐州医学院“振兴计划”项目（2012）;徐州医学院研究生科技创新计划项目（XXYCl3-24）

摘　　要：目的：构建肺癌放射抗性细胞系，观察细胞在形态、凋亡、侵袭迁移能力和上皮间质转化的改变。方法采用小剂量等分割照射法、亚致死剂量照射法及梯度递增照射法筛选肺癌A549和H1299放射抗性细胞系，显微镜下观察细胞形态学改变，克隆形成实验检测放射敏感性，CCK？8法检测细胞受照后存活率；流式细胞仪检测凋亡率；Transwell实验检测细胞侵袭迁移能力的变化；蛋白印迹法检测上皮间质转化标志蛋白表达。结果梯度递增照射法构建肺癌放射抗性细胞系可行性好，得到放射抗性细胞A549R和H1299R，形态学观察示放射抗性细胞向间质细胞形态转变。分别以A549和H1299细胞为对照，A549R和H1299R细胞D0、Dq、SF2增加（P＝0．017和0．033、0．001和0．000、0．000和0．008），α和α／β值减少（ P＝0．018和0．001、0．007和0．009）。 A549R和H1299R细胞在不同照射剂量下存活率均大于对照组（P值均＜0．05），受照后凋亡率降低（P＝0．02和0．01），侵袭率及迁徙率增高（ P＝0．000和0．001及0．001和0．002），E？钙黏蛋白表达下调和波形蛋白表达增高（ P＝0．00和0．01及P＝0．02和0．01）。结论成功构建肺癌放射抗性细胞系，其侵袭迁移能力增强，并且可能与上皮间质转化有关系。Objective To establish radiation？resistant lung carcinoma cell lines, and to investigate the changes in morphology, apoptosis, invasive migration, and epithelial？mesenchymal transition （ EMT） in cells. Methods The radiation？resistant lung carcinoma cell lines were obtained by exposure of lung carcinoma cell lines, A549 and H1299, to radiation with a low dose in fractions, a sublethal dose, or a gradually increasing dose. The morphological changes in cells, radiosensitivity, survival rates after exposure, apoptosis rates, changes in invasive migration, and expression of EMT marker proteins were evaluated using microscopy, colony formation assay, CCK？8 assay, flow cytometry, transwell migration assay, and Western blot, respectively. Results Radiation with a gradually increasing dose successfully induced the radiation？resistant cell lines, A549R and H1299R. The morphological study showed that the morphology of radiation？resistant cells was converted to the morphology of mesenchymal cells. Compared with A549 and H1299 cells, the values of D0 , Dq , and SF2 were significantly increased in A549R （ P=0.017,P=0.001,P=0.000） and H1299R （P=0.033,P=0.000,P=0.008） cells, respectively;the values of α and α/β were significantly reduced in A549R （P=0.018;P=0.007） and H1299R （P=0.001;P=0.009） cells, respectively. The survival rates in A549R and H1299R cells after exposure to radiation with various doses were significantly higher than those in the control groups （all P＆lt;0.05）. After exposure, the apoptosis rates were significantly reduced in A549R and H1299R cells （ P=0.02,P=0.01）;the invasion and migration rates were significantly increased in A549R （P=0.000;P=0.001） and H1299R （P=0.001,P=0.002） cells;the expression of E？cadherin was significantly down？regulated in A549R and H1299R cells （P=0.00,P=0.01）, while the expression of vimentin was significantly elevated in A549R and H1299R cells （ P= 0. 02, P= 0. 01 ） . Conclusions The radiation？resistant lung carcino

关 键 词：肺癌细胞系 放射抗性 克隆形成分析 Transwell细胞分析 上皮间质转化 

分 类 号：R734.2[医药卫生—肿瘤]