饥饿诱导K562/ADM细胞自噬及其与凋亡的关系  

Relationship between the changes in autophagic activity and apoptosis of K562/ADM cells induced by starvation

作　　者：成娟[1] 薛明明 马海珍[1] 张豪[1] 赵龙[1] 

机构地区：[1]兰州大学第一医院血液科,甘肃兰州730000

出　　处：《兰州大学学报（医学版）》2018年第1期53-58,共6页Journal of Lanzhou University（Medical Sciences）

基　　金：甘肃省普通中医药科研项目(甘肃省中医药管理局GZK-2012-14)

摘　　要：目的探讨饥饿诱导人白血病K562/ADM细胞自噬及与凋亡的关系和可能的机制。方法在培养K562/ADM细胞至对数生长期后,以磷酸盐缓冲溶液培养3、6、9 h后收集细胞,透射电镜观察自噬体,流式细胞仪检测单丹磺酰戊二胺(MDC)染色荧光强度,Annexin V/PI双染检测K562/ADM细胞凋亡,实时荧光定量PCR检测LC3、Beclin-1和Bcl-2基因的m RNA变化,Western blot检测LC3、Beclin-1和Bcl-2蛋白。结果饥饿处理3 h后K562/ADM细胞自噬活性明显增强,透射电镜下观察到典型自噬体的形成,其MDC阳性细胞明显增加,LC3基因m RNA的表达增高,LC3-Ⅱ蛋白表达增加,但随着饥饿时间延长,自噬活性却逐渐降低;饥饿促进K562/ADM细胞凋亡,且凋亡率随饥饿时间延长而逐渐增加。饥饿后K562/ADM的Beclin-1表达与自噬活性改变呈正相关,与凋亡呈负相关;Bcl-2表达与自噬活性改变呈负相关,与凋亡呈正相关。结论饥饿诱导白血病细胞K562/ADM发生保护性自噬,抑制其凋亡的发生,Beclin-1与Bcl-2可能参与了自噬抑制凋亡的机制形成。Objective To investigate the relationship between the changes in autophagic activity and apoptosis of K562/ADM cells induced by starvation. Methods Human leukemia K562/ADM cells were induced by star- vation （3 h, 6 h and 9 h） in vitro. Autophagic vacuoles were observed by electron microscope, the apoptotic rates and the increased MDC （monodansylcadaverin）-positive cells of K562/ADM were detected with flow cytometry, the expression of LC3 mRNA, Beclin-1 mRNA and Bcl-2 mRNA were detected by real-time quan- titative PCR （qPCR）, the expression ofLC3, Beclin-1, Bcl-2 protein and the result of LC3-I converted to LC3- II were detected by Western blot. Results Starvation could induce obvious changes in the autophagic activity of K562/ADM cells after their being starved 3 h, obvious autophagic vacuoles were observed under electron microscope, the increased expression of LC3 gene mRNA and LC3- II protein was detected, but autophagic activity of K562/ADM ceils decreased with prolonged starvation. Starvation could induce the increased apop- tosis in a time-dependent manner; the expression of Beclin-1 in starved K562/ADM was positively correlated with the changes of autophagic activity but negatively correlated with apoptosis, while the expression of Bcl-2 was opposite. Conclusion Starvation could induce changes in the autophagic activity, which may protectagainst apoptosis induced by starvation in K562/ADM cells, and Beclin-1 and Bcl-2 may be involved in the molecular mechanisms.

关 键 词：K562/ADM细胞 自噬 凋亡 

分 类 号：R733.71[医药卫生—肿瘤]