低剂量照射对人红白血病细胞株K562凋亡、Bcl-2、Bax及p53蛋白表达影响的实验研究

The in vitro experimental study of influence on Bcl-2,Bax,p53 change and apoptosis with low dose irradiation in leukemia K562

目的探讨低剂量照射及低剂量联合大剂量照射对人红白血病细胞株K562的凋亡Bcl-2、Bax及p53蛋白表达影响及其可能机制。方法实验分成四组,分别为空白对照组、低剂量照射（low dose radiation,LDR）组、大剂量照射（high dose radiation,HDR）组及低剂量联合大剂量（LDR/HDR）组;体外培养K562,取对数生长期细胞分组、分瓶,分别给予不同剂量的6MV-X射线照射;照射后按不同时间点收集细胞,流式细胞仪检测细胞凋亡,Western Blot印迹检测Bcl-xl、Bax及p53蛋白表达变化情况。结果 1LDR照射后48小时凋亡增加（P〈0.05）,96小时达峰值（P〈0.01）,且随照射剂量增大而增加,0.5Gy、0.8Gy剂量组最高（P〈0.01）;LDR联合HDR照射后24小时凋亡增加（P〈0.05）,96-120小时达峰值（P〈0.01）,持续时间较长,0.5Gy与0.8Gy联合组凋亡高,较对照组及HDR组比较差异均有显著性。2LDR照射后24小时Bclxl表达随照射剂量增大而逐渐减少;LDR联合HDR照射各组Bcl-xl蛋白表达量进一步下降,明显低于LDR组及HDR组（P〈0.01）;Bax表达变化与Bcl-xl相反,LDR各组随照射剂量增大而增加（P〈0.01）,LDR联合HDR各组Bax表达量进一步增加,明显高于LDR组及HDR组,差异有显著性（P〈0.01）;p53蛋白表达在LDR或LDR联合HDR照射24小时均无明显变化（P〉0.05）。结论 LDR能诱导K562凋亡,LDR联合HDR能增强HDR对K562的凋亡作用,其机制与LDR辐射超敏感性有关,通过下调Bcl-xl表达及上调Bax表达,启动非p53依赖凋亡途径;但所需诱导照射剂量较大,且p53蛋白表达无明显变化,可能与K562高表达Bcl-xl及p53突变等有关。

Objective To investigate the effect on expression change of Bcl-2, Bax, p53 and apoptosis by low dose radiation or low dose radiation combining high dose radiation in human erythro-leukemia cell line K562 in vitro and to reveal the possible mechanisms of apoptosis and hyper-sensitivity which induced by low dose radiation. Method The test were divided into four groups, including blank control group,low dose radiation group（ LDR groups） ,high dose radiation group （ HDR groups ） and low dose radiation combining high dose radiation group （ LDR/HDR groups） . The erythro-leukemia cell K562 were cultivated in vitro. Cells were radiation at room temperature with 6-MV X-ray by linear accelerator. After irradiation,radiated cells were harvested for detection of apoptosisin flow cytometer （FCM）;Western Blotting were performed to detect the expression of Bcl-xl,Bax and p53. Result ①The percentages of apoptosis in the LDR group after radiation 48 hours was raising lasting to 96 hours, especially in 0. 5Gy and 0. 8Gy group （P〈0. 01）. The number of apoptosis cells in the LDR combining HDR group were increasing from 24 hours to120 hours after radiation, during 96 hours to 120 hours,the number of apoptosis cells arrive the peak.② Compared with blank control group,the expression of Bax protein were up-regulated significantly and the quantity of Bax was dependent on the radiation doses after radiation 24 hours. The expression of Bax protein in LDR combining HDR group increased significantly than in LDR group. However, the expression of Bcl-xl protein were contrast to the Bax, down-regulated significantly in all radiation groups and the quantity of Bcl-xl protein was also dependent on the radiation doses. The expression of p53 protein were no obviously change in LDR groups, HDR group and LDR combining HDR groups. Conclusion LDR may induce the K562 apoptosis and enhance the effect of HDR induced K562 apoptosis. LDR may induce the K562 cells up-regulated Bax protein expression and down-regulated Bcl-xl protein expression.