PC12细胞氧糖剥夺/再灌注损伤中自噬与凋亡现象  

Autophagy and Apoptosis in PC12 Cells Injury Induced by Oxygen and Glucose Deprivation/ Reperfusion

作　　者：董琳[1,2] 童煜[2] 毛萌[1,2,3] 

机构地区：[1]四川大学华西第二医院儿科,成都610041 [2]四川大学华西第二医院西部妇幼医学研究院早期发育与损伤实验室,成都610041 [3]妇儿疾病与出生缺陷教育部重点实验室,成都610041

出　　处：《实用儿科临床杂志》2012年第18期1397-1401,共5页Journal of Applied Clinical Pediatrics

基　　金：国家自然科学基金(30973215);长江学者和创新团队发展计划(IRT0935)

摘　　要：目的观察氧糖剥夺/再灌注(OGD/OGD-R)诱导大鼠嗜铬细胞瘤细胞PC12细胞自噬和凋亡的发生及进展。方法PC12细胞经氧糖剥夺及再灌注处理建立OGD/OGD-R模型,在处理不同时间点,应用流式细胞仪检测OGD组及OGD-R组细胞活性和凋亡率;Western blot法检测自噬相关蛋白LC3、Beclin 1表达;免疫荧光法观察自噬小体;分别使用自噬上下游抑制剂(3-甲基腺嘌呤、E64 d+pepstatin A)进行调控,观察自噬的波动。结果随着氧糖剥夺时间的延长,OGD组细胞形态损伤逐步加重,凋亡率显著增高,呈时间依赖性;OGD-R组早期细胞活性略有恢复,随着再灌注时程的延长,细胞凋亡率逐渐增高。自噬相关蛋白LC3Ⅱ在OGD短暂处理后其表达即有上升,3 h左右达到峰值,6～8 h恢复到基础水平;Beclin 1蛋白呈先上升后平稳下降趋势。OGD 3 h时间点诱导自噬合并3-甲基腺嘌呤处理后自噬体减少明显;而E64 d+pepstatin A可导致LC3Ⅱ的积聚。OGD-R阶段LC3Ⅱ进一步升高至再灌注12 h后开始下降,Beclin 1蛋白表达呈升高趋势并持续至再灌注24 h。结论 OGD/OGD-R均能激活PC12细胞自噬与凋亡,可为探索自噬与凋亡间潜在的串流奠定基础。Objective To observe the effects of autophagy and apoptosis in oxygen and glucose deprivation/reperfusion（ OGD/OGD - R） - induced injury model of rat adrenal medullary pheochromocytoma cell line PC12. Methods The model of OGD/OGD - R injury on PC12 cells was established. The cell viability and apoptosis rate were analyzed using flow cytometry, and Western blot assay was used to evaluate the expression of LC3 and Beclin 1 proteins in OGD group and OGD - R group at different time. LC3 immunofluorescence staining was used to detect autophagosome. 3 - methyladenine, E64 d and pepstatiu A, upstream and downstream inhibitors of autophagy, were separately added to assess the fluctuation of autophagy level. Results PC12 cell morphology damage aggravated and apoptosis percentage increased in OGD time - dependence. The cell viability in OGD - R group recovered somewhat in the early phase, while over time, apoptosis percentage gradually increased. LC3 II protein expression up - regulated briefly,peaked at 3 h and returned to baseline at 6 -8 h. The variation tendency of Beelin 1 a/se increased, followed by a slight decline. 3 - mcthyladenine inhibited autophagy induced by OGD 3 h with dominant decrease of autophagosome formation. The combination treatment of E64 d and pepstatin A blocked degradation pathways of autophagosome and the a- mount of LC3 II accumulated markedly. In the reperfusion phase ,the expression of LC3 II further enhanced ,and reached its peak at 12 h and then began to reduce ; Beclin 1 expression continued trending higher and lasted till 24 h. Conclusions Both OGD and OGD - R injury can activate apoptosis and autophagy pathways with varying degrees in PC12 cells. This may lay the foundation for the further exploration of the potential streaming between the 2 mechanisms.

关 键 词：自噬 凋亡 PC12 氧糖剥夺 再灌注 

分 类 号：R363[医药卫生—病理学]