永久缺血缺氧对PC12细胞自噬的影响

发布时间：2019-07-06 20:48

【摘要】：目的建立缺血缺氧损伤细胞模型,探讨永久性缺血缺氧致神经细胞损伤机制及其对PC12细胞自噬的影响。方法采用经典的神经细胞模型PC12细胞作为研究对象,利用无糖的DMEM培养基和缺氧罐(95%N_2和5%CO_2)培养PC12细胞,模拟体内神经细胞缺血缺氧环境。细胞分为对照组(在正常条件下,使用完全培养基培养细胞)和糖氧剥夺(OGD)处理组(在缺氧条件下,使用OGD培养基培养细胞):0.5h(OGD+0.5h)、2h(OGD+2h)、6 h(OGD+6h)、12h(OGD+12h)和24h(OGD+24h)。利用MTT检测细胞存活率,通过流式细胞仪检测细胞凋亡率,比色法检测细胞乳酸脱氢酶(LDH)释放率,免疫荧光以及Western blotting法检测低氧诱导因子-1α(HIF-1α)、环氧化酶-2(COX2)、微管相关蛋白轻链3(LC3)和Beclin-1的表达,透射电子显微术检测自噬体的超微结构,探讨不同永久性缺血缺氧时间对细胞损伤以及自噬的影响。结果与对照组相比,细胞存活率下降,呈OGD时间依赖性,且自OGD 6h显著下降,差异具有统计学意义(P0.05);细胞凋亡率和坏死率持续增高,与OGD时间呈正相关,且自OGD 12h后显著升高,差异具有统计学意义(P0.05);HIF-1α和COX2蛋白表达随OGD时间延长逐渐升高,OGD 12h后增高显著,差异具有统计学意义(P0.05)。OGD组被荧光标记的绿色的LC3蛋白相对于对照组,数量增多,绿色荧光增强。Beclin-1蛋白表达结果显示,Beclin-1的表达与OGD时间呈正相关,并由OGD6h开始明显增加,差异具有统计学意义(P0.05)。结论缺血缺氧能够诱导PC12细胞氧化应激及自噬激活。
[Abstract]:Objective to establish a cell model of ischemia and hypoxia injury and to explore the mechanism of nerve cell injury induced by permanent ischemia and hypoxia and its effect on autophagy of PC12 cells. Methods the classical nerve cell model PC12 cells were used as the research object. PC12 cells were cultured in sugar-free DMEM medium and hypoxia tank (95%N_2 and 5%CO_2) to simulate the ischemic and anoxic environment of nerve cells in vivo. The cells were divided into two groups: control group (cultured in complete medium under normal conditions) and glucose-oxygen deprived (OGD) group (cultured in OGD medium under hypoxia): 0.5h (OGD 0.5h), 2h (OGD 2h), 6 h (OGD 6 h), 12 h (OGD 12 h) and 24 h (OGD 24 h). The cell survival rate was detected by MTT, the apoptosis rate was detected by flow cytometry, the release rate of lactic dehydrogenase (LDH) was detected by colorimetric assay, the expression of hypoxia inducible factor-1 伪 (HIF-1 伪), cyclooxygenase-2 (COX2), microtubule-associated protein light chain 3 (LC3) and Beclin-1 were detected by immunofluorescence and Western blotting, and the ultrastructure of autophagy was detected by transmission electron microscopy. To investigate the effects of different permanent ischemia and hypoxia time on cell injury and autophagy. Results compared with the control group, the cell survival rate decreased in a OGD time-dependent manner, and decreased significantly since OGD 6 h, the difference was statistically significant (P 0.05), the apoptosis rate and death rate continued to increase, which was positively correlated with OGD time, and increased significantly after 12 h OGD, the difference was statistically significant (P 0.05). The expression of HIF-1 伪 and COX2 protein increased gradually with the prolongation of OGD time, and increased significantly after 12 hours of OGD, the difference was statistically significant (P 0.05). Compared with the control group, the number of green LC3 protein labeled with fluorescence increased and the green fluorescence increased. Beclin-1 protein expression showed that the expression of Beclin-1 was positively correlated with OGD time, and began to increase significantly by OGD6h, the difference was statistically significant (P 0.05). Conclusion ischemia and hypoxia can induce oxidative stress and autophagy activation in PC12 cells.
【作者单位】：河南大学神经生物学研究所;河南大学医学院生物化学与分子生物学教研室;河南大学护理学院;
【基金】：国家自然科学基金(31070952,U1204311)
【分类号】：R743.3

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