摘要
目的探究不同时长氧糖剥夺(OGD)后小鼠皮层神经元中线粒体自噬相关蛋白PTEN诱导的假定激酶1(PINK1)表达的时空动态改变。方法 C57BL/6J小鼠皮层神经元原代培养7 d,以OGD不同时长(0 h、2 h、4 h、6 h、8 h)作为观察时间点。倒置显微镜下观察神经元形态变化;MTT比色法检测神经元存活率;蛋白印迹法(Western blot,WB)观察LC3-Ⅱ、PINK1和Parkin表达的时间变化;免疫荧光观察PINK1OGD后表达的空间改变。结果 OGD严重损伤神经元活性,且随着时间延长细胞活性呈进展性下降趋势;OGD后自噬标志性分子LC3-Ⅱ和PINK1开始升高(4 h达到峰值)(P<0.05),后进行性下降,而Parkin表达逐渐下降(P<0.05);PINK1在胞浆可见均匀表达,在OGD后荧光强度短暂增高,并随OGD时间逐渐减弱。结论不同时长OGD可造成不同程度神经元损伤,并激活PINK1介导的线粒体自噬,PINK1可能通过影响线粒体在脑缺血损伤的病理生理过程中发挥作用。
Objective The temporal and spatial expression changes of mitochondrial autophagy related proteinPTEN induced putative kinase 1（PINK1） at different time points after oxygen-glucose deprivation（OGD） in mouse cortical neurons were investigated. Methods Primary cortical neurons derived from C57 BL/6 J mouse were cultured for 7 d and then different time points（2 h/4 h/6 h/8 h） after OGD were used as set points. Morphological change was detected by inverted microscope; cell viability was detected by MTT; temporal variations of LC3,PINK1,Parkin was detected by Western blot; spatial variations of the expression of PINK1 was detected by immunofluorescence.Results The cell viability was impaired by OGD,and aggravated as the duration of OGD increased. The expression of LC3-Ⅱand PINK1 peaked at 4 h after OGD（P〈0. 05）,and the expression was gradually decreased since then.Parkin was decreased meanwhile（ P〈0. 05）. PINK1 was evenly expressed in cytoplasm and the intensity of immunofluorescence was temporally increased after OGD and was gradually decreased after that. Conclusion Different periods of OGD treatment induce the neuronal damage at different levels,and subsequently activate the PINK1-mediated mitochondrial autophagy,which may play a role in the pathophysiological process of cerebral ischemia via mitophagy.
作者
王元欣
张磊
黑悦
鱼洋
岳康异
武秀权
蒋晓帆
WANG Yuanxin;ZHANG Lei;HEI Yue;YU Yang;YUE Kangyi;WU Xiuquan;JIANG Xiaofan(Department of Neurosurgery, Xijing Hospital, Air Force Military Medical University, Xi＇an 710032;Department of Neurosurgery, Fufeng County People＇s Hospital, Baofi 722200, China)
出处
《中华神经外科疾病研究杂志》
CAS
2018年第2期143-146,共4页
Chinese Journal of Neurosurgical Disease Research
基金
国家自然科学基金资助项目(81671303)
