中文摘要：

目的 通过检测人胚肾上皮细胞HEK293受不同剂量α粒子照射后,src激酶活性和细胞自噬系统的变化,探讨调控细胞自噬对高剂量辐射诱导细胞死亡的影响.方法 将人胚肾上皮细胞HEK293分为3组：0 cGy（对照）组、241Am α粒子照射低剂量（10 cGy）组和高剂量（300 cGy）组.应用免疫印迹实验检测细胞内源性蛋白LC3Ⅰ/Ⅱ和src激酶的变化;分子探针检测细胞内活性氧（ROS）水平;用ROS淬灭剂DMSO预处理照射细胞,并用免疫印迹法检测照射后src激酶的变化;使用自噬诱导剂雷帕霉素预处理照射细胞,以PI染色流式细胞术测定细胞死亡率.结果 与0 cGy相比,10 cGy α射线照射后LC3 Ⅰ/Ⅱ比例降低（=4.07,P＜0.05）,胞质内具有绿色荧光点状GFP-LC3的细胞比例升高（t=12.29,P＜0.05）,自噬被诱导;而300 cGy照射后,LC3Ⅰ/Ⅱ比例升高（t=2.93,P＜0.05）,胞质内GFP-LC3形态无明显变化,自噬被抑制.与0 cGy相比,10和300 cGy照射后4h均能提升细胞内ROS水平（t=17.93、22.88,P＜0.05）,且300 cGy比10 cGy照射诱发的ROS更多（t=15.76、22.66、14.22,P＜0.05）.与0 cGy相比,10 cGy照射使src激酶419位酪氨酸磷酸化水平升高（t=5.66,P＜0.05）,而300 cGy照射则降低其磷酸化水平（=4.67,P＜0.05）.DMSO能够部分逆转高低剂量照射对src激酶活性的影响.辐照前以自噬诱导剂雷帕霉素处理细胞则能降低300 cGy照射诱发的细胞死亡率（t=12.14,P＜0.05）.结论 高低剂量α粒子分别抑制和激活src激酶以及细胞自噬,ROS参与介导辐照对src激酶活性及自噬系统的影响.对自噬系统的干预能够降低细胞的辐射敏感性.

英文摘要：

Objective To investigate the modulation role of autophagy in radiation-induced cell death by detecting the response of src kinase activity and autophagy in HEK293 cells irradiated with different dose of α-particle.Methods HEK293 cells were irradiated by contral group （0 cGy） a low dose group （10 cGy） and high dose group （300 cGy） α-particles.Molecular probe 2＆#39;,7＆#39;-dichlorofluorescin （DCFHDA） was used to detect the cell ROS.The src kinase activity and endogenous protein level of LC3Ⅰ/Ⅱ were monitored by Western Blot.Cell death rate of irradiated cells pretreated with autophagy inducer of rapamycin was tested by flow cytometry.Results Compared with control group,the ratio of LC3Ⅰ/Ⅱ decreased （t =4.07,P 〈 0.05） and the percentage of cells with GFP-LC3 punctuate dots increased （t =12.29,P 〈0.05） under 10 cGy irradiation,indicating the induction of autophagy.On the contrary,the ratio of LC3Ⅰ/Ⅱ increased （t =2.93,P 〈 0.05） and the GFP-LC3 morphology had no obvious change under 300 cGy irradiation.The cellular ROS level reached to the maximum value at 4 h postirradiation.Both 10 cGy and 300 cGy irradiation could elevate the ROS level （t =17.93,22.88,P 〈0.05）,whereas the amplitude of elevation of 300 cGy irradiation was higher than that of 10 cGy irradiation （t =15.76,22.66,14.22,P 〈 0.05）.Compared with control group,the 419th site of tyrosine residue in src kinase manifested hyper-phosphorylation （t =5.66,P 〈0.05） under 10 cGy irradiation whereas it had hypo-phosphorylation under 300 cGy irradiation （t =4.67,P 〈 0.05）.Treatment of cells with DMSO could partly restore the impact on src kinase activity under high or low dose irradiation.Pre-treating the cells with autophagy inducer rapamycin could reduce cell death under 300 cGy irradiations （t =12.14,P 〈 0.05）.Conclusions High or low dose of α-particles irradiation could inhibit or activate src kinase and autophagy system,respectively.ROS mediated the response of src kinase activity