永久缺血缺氧对PC12细胞自噬的影响

doi:10.16098/j.issn.0529-1356.2017.03.001

摘要/Abstract

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% N₂和5% CO₂）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)、6h（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（P<0.05）；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（P<0.05）；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（P<0.05）。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OGD 6h开始明显增加，差异具有统计学意义（P<0.05）。结论缺血缺氧能够诱导PC12细胞氧化应激及自噬激活。

关键词: 缺血, 缺氧, 糖氧剥夺, 氧化应激, 免疫印迹法, PC12细胞

Abstract:

Objective To investigate the mechanism of permanent injuries induced by oxygen glucose deprivation treatment and the effect of these injuries on autophagy using the cell model of ischemic and hypoxic injuries. Methods In the present study, PC12 cells were used. To establish the oxygen-glucose deprivation (OGD) model, PC12 cells were cultured with glucose free DMEM medium under the condition of 95% N₂ and 5% CO₂， to imitate the permanent ischemia hypoxia (IH) injuries in vivo. The cells were divided into（1）control group: the cell culture in normal medium with normoxia;（2）OGD groups: the cells were cultured in OGD medium for different times from 0.5 hour to 24 hours, such as OGD 0.5 hour (OGD+0.5 hour), 2 hours（OGD+2 hours), 6 hours（OGD+6 hours), 12 hours（OGD+12 hours), 24 hours（OGD+24 hours) groups. The cell viability and cell apoptosis rate were analyzed with MTT assay and flow cytometry. Lactate dehydrogenase (LDH) release level was tested with the colorimetric method. The expressions of hypoxia inducible factor 1α (HIF-1α, a key regulator in hypoxia), cyclooxygenase 2 (COX2, inflammatory indicator), microtubule-associated protein 1 light chain 3（LC3）and Beclin-1 were examined and analyzed with immunocytochemistry and Western blotting. The ultrastructure of autophagosomes were examined by transmission electron microscopy. The effect of different OGD times on ischemic and hypoxic injuries and autophagy was explored. Results The cell viability decreased in OGD groups with time prolongation, compared with control group, the decrease was significant and difference appeared a statistical significance after 6 hours OGD (P<0.05). Cell apoptosis and necrosis rates showed positive correlation with OGD time prolongation, and significant difference appeared after 12 hours OGD (P<0.05). The results showed that the expression of HIF-1α and COX2 were up-regulated in OGD groups with time prolongation, and significant difference appeared after 12 hours OGD (P<0.05). The LC3 was labelled by green-fluorescent protein in OGD groups with number increasing, and fluorescence intensity enhancement. The results of Western blotting revealed that Beclin-1 was positive relevant with OGD time, and significant difference appeared after 6 hours OGD (P<0.05). Conclusion Oxygen glucose deprivation can induce PC12 cells oxidative stress response and autophagy activation.

Key words: Ischemia, Hypoxia, Oxygen-glucose deprivation, Oxidative stress response, Western blotting, PC12 cell

厉永强王萌王来刘彬石贞玉邓锦波. 永久缺血缺氧对PC12细胞自噬的影响[J]. 解剖学报, 2017, 48(3): 242-253.

LI Yong-qiang WANG Meng WANG Lai LIU Bin SHI Zhen-yu DENG Jin-bo. Effects of permanent ischemia and hypoxia on PC12 cells autophagy[J]. Acta Anatomica Sinica, 2017, 48(3): 242-253.

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