饥饿对Beclin-1依赖的Ana-1细胞自噬和凋亡的影响

目的：研究饥饿对Ana-1细胞自噬和凋亡的影响，并探讨其作用机制。方法：体外培养Ana-1细胞，将细胞分为对照组（饥饿0h）和饥饿3、6、9、12、24h组（饥饿组），观察饥饿对细胞自噬与凋亡的影响；将Ana-1细胞分为空白对照组、饥饿组、3-甲基腺嘌呤（3-MA）组、3-MA联合饥饿组，孵育24 h，采用Western blotting法检测各组细胞中自噬相关蛋白LC3-Ⅱ/LC3-Ⅰ比值及Beclin-1蛋白表达水平和凋亡相关蛋白Caspase-3蛋白水平，采用倒置显微镜观察饥饿6、12和24 h组细胞形态表现。结果：与对照组比较，饥饿6、12和24h组Ana-1细胞中LC3-Ⅱ/LC3-Ⅰ比值，饥饿3、6、9、12和24 h组Ana-1细胞中Beclin-1蛋白表达水平均明显升高（P<0.05或P<0.01），饥饿3、6、9、12和24 h组Ana-1细胞中Caspase-3水平明显降低（P<0.01），呈时间依赖性；倒置显微镜下观察，与对照组比较，不同时间饥饿组细胞形态发生改变，细胞皱缩、出现碎片，随着时间的延长，细胞变化更加明显。与饥饿组比较，3-MA联合饥饿组Ana-1细胞中LC3-Ⅱ/LC3-Ⅰ比值和Beclin-1蛋白表达水平及Caspase-3蛋白水平均明显降低（P<0.05或P<0.01）。结论：饥饿可诱导Beclin-1依赖的Ana-1细胞自噬并引起细胞凋亡。

Effects of starvation on autophagy and apoptosis of Beclin-1-dependent Ana-1 cells

Objective:To study the effects of starvation on the autophagy and apoptosis of Ana-1 cells,and to explore their mechanisms. Methods:The Ana-1 cells were cultured in vitro and starved for 0 h(control group),3,6,9,12 and 24 h(starvation group)to study the effects of starvation on the autophagy and apoptosis.The Ana-1 cells were divided into blank control group,starvation group,3-methyladenine group(3-MA) and 3-MAcombined with starvation group,and they were incubated for 24 h to explore their mechanisms.The ratio of autophagy associated protein LC3-Ⅱ/LC3-Ⅰ,the expression levels of Beclin-1 and apoptosis-related protein Caspase-3 in the cells in various groups were detected by Western blotting method.Cell morphology 6,12,and 24 h after starvation was observed by inverted microscope. Results:Compared with control group,the ratio of LC3-Ⅱ/LC3-Ⅰ in the Ana-1 cells in starvation group(6,12,and 24 h)and the expression levels of Beclin-1 in starvation group(3,6,9,12,and 24 h) were significantly increased(P<0.05 or P<0.01),and the Caspase-3 levels in starvation group(3,6,9,12,and 24 h) were decreased significantly(P<0.05 or P<0.01) in a time-dependent manner.Compared with control group,the cells in starvation group at different time were changed in shape:the cells shrank,the fragments appeared,and the changes of cells became more obvious with the prolongation of time.Compared with starvation group,the ratio of LC3-Ⅱ/LC3-Ⅰ and the expression levels of Beclin-1 and Caspase-3 in the Ana-1 cells in 3-MA combined with starvation group were decreased significantly(P<0.05 or P<0.01). Conclusion:Starvation could induce the autophagy and apoptosis of the Beclin-1-dependent Ana-1 cells.