CAJ | 학술논문

目的观察A549细胞的低剂量辐射超敏感性现象,探讨其发生的机制.方法 A549细胞接受0～2 Gy的60Co γ射线照射后,流式细胞仪对其分选计数,克隆形成法检测细胞存活分数,Western blot法检测ATMl981Ser-P蛋白表达,Hoechst 33258荧光染色法、AnnexinV-FITC/PI双染流式细胞仪检测细胞凋亡,PI单染流式细胞仪检测细胞周期.结果细胞在0～0.3 Gy表现出单位剂量杀伤增强,在0.3～0.5 Gy表现出一定的辐射抗性,0.5 Gy后的区域存活分数随辐射剂量的增加而降低.照射后1 h,ATM激酶在0.2 Gy时开始活化,0.5 Gy时活化达高峰(t=7.96,P＜0.05);与0.5 Gy相比1.0和2.0 Gy的活化水平无明显变化(t=0.69、0.55,P＞0.05).照射后24 h,部分细胞发生凋亡,其凋亡曲线与存活曲线相吻合.与未照射组相比,0.1和0.2 Gy组在各时间点(照射后6、12和24 h)的细胞周期无明显变化,而0.3、0.4和0.5 Gy组,照射后6和12 h细胞发生G2/M期阻滞(t=2.87、2.88、4.92和3.70、3.12、8.11,P＜0.05),照射后24 h G2/M期细胞比例下降(t=3.87、4.77、3.01,P＜0.05).结论 A549细胞存在HRS/IRR现象,其发生可能与ATM激酶、细胞周期变化有关,凋亡是细胞死亡的主要方式.
목적 관찰A549세포적저제량복사초민감성현상,탐토기발생적궤제.방법 A549세포접수0～2 Gy적60Co γ사선조사후,류식세포의대기분선계수,극륭형성법검측세포존활분수,Western blot법검측ATMl981Ser-P단백표체,Hoechst 33258형광염색법、AnnexinV-FITC/PI쌍염류식세포의검측세포조망,PI단염류식세포의검측세포주기.결과 세포재0～0.3 Gy표현출단위제량살상증강,재0.3～0.5 Gy표현출일정적복사항성,0.5 Gy후적구역존활분수수복사제량적증가이강저.조사후1 h,ATM격매재0.2 Gy시개시활화,0.5 Gy시활화체고봉(t=7.96,P＜0.05);여0.5 Gy상비1.0화2.0 Gy적활화수평무명현변화(t=0.69、0.55,P＞0.05).조사후24 h,부분세포발생조망,기조망곡선여존활곡선상문합.여미조사조상비,0.1화0.2 Gy조재각시간점(조사후6、12화24 h)적세포주기무명현변화,이0.3、0.4화0.5 Gy조,조사후6화12 h세포발생G2/M기조체(t=2.87、2.88、4.92화3.70、3.12、8.11,P＜0.05),조사후24 h G2/M기세포비례하강(t=3.87、4.77、3.01,P＜0.05).결론 A549세포존재HRS/IRR현상,기발생가능여ATM격매、세포주기변화유관,조망시세포사망적주요방식.
Objective To study the low dose hyper-radiosensitivity in human lung cancer cell line A549,and its possible mechanisms.Methods Exponentially growing A549 cells were irradiated with 60Co γ-rays at doses of 0-2 Gy.Together with flow cytometry for precise cell sorting,cell survival fraction was measured by mean of conventional colony-formation assay.ATM1981 Ser-P protein expression was examined by Western blot.Apoptosis was identified by Hoechst 33258 fluorescent staining,and Annexin V-FITC and propidium iodide staining flow cytometry.Cell cycle distribution was observed by flow cytometry.Results There was an excessive cell killing per unit dose when the doses were below about 0.3 Gy,and the cells exhibited more resistant response at the doses between 0.3 and 0.5 Gy,the cell survival fraction was decreased as the doses over 0.5 Gy.The expression of ATM1981Ser-P protein was first observed at 0.2 Gy,followed by an increase over 0.2 Gy,and reached the peak at 0.5 Gy(compared with 0.2 Gy group,t=7.96,P＜0.05),with no further increase as the doses at 1.0 and 2.0 Gy(t=0.69,0.55,P＞0.05).24 hours after irradiation,part cells presented the characteristic morpholos4cal change of apoptosis,and the apoptosis curve was coincident with the dose-survival curve.Compared with the control group,the cell cycle had no change post-irradiation to 0.1 and 0.2 Gy.G2/M phase arrest was manifested at 6 and 12 hours post-irradiation to 0.3,0.4 and 0.5 Gy(t=2.87,2.88,4.92 and 3.70,3.12,8.11,P＜0.05),and the ratio of G2/M phase was decreased at 24 hours post-irradiation(t=3.87,4.77,3.01,P＜0.05).Conclusions A549 cells displays the phenomenon of hyper-radiosensitivity(HRS)/induced radioresistance(IRR).The model of cell death induced by low dose irradiation is mainly apoptosis.The activity of ATM and cell cycle change might play an important role in HRS/IRR.