&nbsp;缺氧缺糖/复氧复糖HT22细胞损伤的动态观察

张怡 周晓红 靳晓飞 张颖 高维娟*

doi:10.16098/j.issn.0529-1356.2019.01.003

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解剖学报 ›› 2019, Vol. 50 ›› Issue (1) : 13-17. DOI: 10.16098/j.issn.0529-1356.2019.01.003

神经生物学

缺氧缺糖/复氧复糖HT22细胞损伤的动态观察

张怡周晓红靳晓飞张颖高维娟*

作者信息 +

Dynamic observation of injury of HT22 cell after oxygen and glucose deprivation/reoxygenation

ZHANG Yi ZHOU Xiao-hong JIN Xiao-fei ZHANG Ying GAO Wei-juan*

Author information +

文章历史 +

摘要

目的探讨缺氧缺糖/复氧复糖不同时间点小鼠海马神经元细胞系HT22细胞损伤情况。方法取对数生长期的HT22细胞，随机分为6组：正常对照组、缺氧缺糖/复氧复糖（OGD/R）0 h组（OGD/R 0 h）、缺氧缺糖/复氧复糖12 h组（OGD/R 12 h）、缺氧缺糖/复氧复糖24 h组（OGD/R 24 h）、缺氧缺糖/复氧复糖48 h组（OGD/R 48 h）、缺氧缺糖/复氧复糖72 h组（OGD/R 72 h）。除正常对照组外，其余各组细胞均在缺氧缺糖6 h后进行复氧复糖。倒置显微镜下观察细胞形态，细胞计数试剂盒8（CCK-8）法检测细胞存活率，乳酸脱氢酶（LDH）法检测细胞损伤情况，Bax、Bcl-2免疫荧光染色检测细胞凋亡情况。结果正常组HT22细胞呈两极或多极，突起明显，突起之间相互交织成网状，细胞膜光滑且完整，胞体折光性强，OGD/R各组细胞胞体轴突减少，细胞皱缩，胞质凝聚。与正常组比较，OGD/R各组细胞存活率均显著降低（P<0.05），OGD/R 12 h至OGD/R 24 h达到最低（P<0.05）；OGD/R 12 h及OGD/R 24 h 组LDH显著升高（P<0.05），OGD/R 0 h、48 h、72 h组均有升高趋势。除OGD/R 0 h组和OGD/R 72 h组外，OGD/R 各组Bax/Bcl-2比值均较正常组显著升高（P<0.05），峰值出现于OGD/R 24 h。结论缺氧缺糖/复氧复糖细胞损伤是一个复杂的动态过程，随着复氧复糖时间的延长，细胞损伤不断加重，24 h达到顶峰，随后细胞损伤情况逐渐缓解。

Abstract

Objective To investigate the damage of hippocampal neuron cell line HT22 at different time points after oxygen and glucose deprivation/reoxygenation. Methods Logarithmic growth phase of HT22 cells was randomly divided into six groups, normal control group,OGD/R 0 hour, OGD/R 12 hours, OGD/R 24 hours, OGD/R 48 hours,OGD/R 72 hours groups. All groups were treated with reoxygenation after deprived of oxygen and glucose for 6 hours except normal group.The cell morphology was observed by invertded microscope. Cell counting kit-8(CCK-8) method was used to test the cell survival rate. Lactate dehydrogenase (LDH) method was used to detect cell damage. Bax and Bcl-2 immunofluorescence were used to detect apoptosis. Results HT22 cells in normal group were bipolar or multipolar. Obvious synapses and multiple synapses were woven into a network. Cell membrane was smooth and complete and the refractivity of cell was strong. But the axons decreased, cell was shrinkage and cytoplasmic aggregation in HT22 in OGD/R groups, and the cell viability was significantly reduced（P<0.05）, the lowest point appeared in OGD/R 12 hours to OGD/R 24 hours; OGD/R 12 hours and OGD/R 24 hours LDH increased significantly (P<0.05), OGD/R 0 hour, 48 hours, and 72 hours increased. The ratios of Bax/Bcl-2 in OGD/R groups were significantly increased (P<0.05) except for OGD/R 0 hour and OGD/R 72 hours, and peaked at OGD/R 24 hours. Conclusion Injury of cells afer OGD/R is a complex and dynamic process. Cell injury is aggravating as the reoxygenation time increased and reached a peak in OGD/R 24 hours, from then on, cell damage gradually decreased.

导出引用

张怡周晓红靳晓飞张颖高维娟. 缺氧缺糖/复氧复糖HT22细胞损伤的动态观察[J]. 解剖学报. 2019, 50(1): 13-17 https://doi.org/10.16098/j.issn.0529-1356.2019.01.003

ZHANG Yi ZHOU Xiao-hong JIN Xiao-fei ZHANG Ying GAO Wei-juan. Dynamic observation of injury of HT22 cell after oxygen and glucose deprivation/reoxygenation[J]. Acta Anatomica Sinica. 2019, 50(1): 13-17 https://doi.org/10.16098/j.issn.0529-1356.2019.01.003

参考文献

［1］ National Cardiovascular Center. Report on Cardiovascular Disease in China ［M］. Beijing: Encyclopedia of China Publishing House, 2014:96-97. (in Chinese)

国家心血管病中心. 中国心血管病报告［M］. 北京：中国大百科全书出版社, 2014：96-97.

［2］ Piironen K, Tiainen M, Mustanoja S, et al. Mild hypothermia after intravenous thrombolysis in patients with acute stroke: a randomized controlled trial［J］. Stroke, 2014, 45(2):486-491.

[3］ Bennett DA, Krishnamurthi RV, Barkercollo S, et al. The global burden of ischemic stroke: findings of the GBD 2010 study［J］. Glob Heart, 2014, 9(1):107-112.

［4］ Khandelwal P, Yavagal DR, Sacco RL. Acute Ischemic Stroke Intervention［J］. J Am Coll Cardiol, 2016, 67(22):2631-2644.

［5］ Kotl?ga D, Golab-Janowska M, Masztalewicz M, et al. The emotional stress and risk of ischemic stroke［J］. Neurol Neurochir Pol, 2016, 50(4):265-270.

［6］ Hou YC, liou KT, Chen CM, et al. Preventive effect of silymarin in cerebral ischemia-reperfusion-induced brain injury in rats possibly through impairing NF-κB and STAT-1 activation［J］.Phytomedicine，2010,17(12):963-973.

［7］ Wang J, Han D, Sun M, et al. Cerebral ischemic post-conditioning induces autophagy inhibition and a HMGB1 secretion attenuation feedback loop to protect against ischemia reperfusion injury in an oxygen glucose deprivation cellular model ［J］. Mol Med Rep, 2016, 14(5):4162-4172.

［8］ Jin XF, Zhang Y, Zhou XH, et al. Effects of astragaloside Ⅳ on apoptosis of PC12 cells induced by hypoxia/hypoglycemia and reoxygenation ［J］.Chinese Pharmacological Bulletin, 2016, 32 (10):1411-1415.(in Chinese)

靳晓飞, 张颖, 周晓红,等. 黄芪甲苷对缺氧缺糖/复氧复糖PC12细胞凋亡的影响［J］. 中国药理学通报, 2016, 32(10):1411-1415.

［9］ Taylor RC, Cullen SP, Martin SJ. Apoptosis: controlled demolition at the cellular level［J］. Nat Rev Mol Cell Biol, 2008, 9(3):231-241.

［10］ Kalinichenko SG, Niu M. Morphological characteristic of apoptosis and its significance in neurogenesis ［J］. Morfologiia, 2007, 131(2):16-28.

［11］ Song XY, Hu JF, Chen NH. Neurons apoptosis and cerebral ischemia［J］.Chinese Pharmacological Bulletin,2012,28(3):307-310. (in Chinese)

宋修云, 胡金凤, 陈乃宏. 神经细胞凋亡与脑缺血疾病［J］. 中国药理学通报, 2012, 28(3):307-310.

［12］ Pohl SO, Agostino M, Dharmarajan A, et al. Crosstalk between cellular redox state and the anti-apoptotic protein Bcl-2［J］. Antioxid Redox Signal, 2018,29(13):1215-1236.

［13］ Loor G, Kondapalli J, Iwase H, et al. Mitochondrial oxidant stress triggers cell death in simulated ischemia-reperfusion ［J］. Biochim Biophys Acta, 2011, 1813(7):1382-1394.

［14］ Kristen AV, Ackermann K, Buss S, et al. Inhibition of apoptosis by the intrinsic but not the extrinsic apoptotic pathway in myocardial ischemia-reperfusion［J］. Cardiovasc Pathol, 2013, 22(4):280-286.

［15］ Liu QS, Deng R, Li S, et al. Ellagic acid protects against neuron damage in ischemic stroke through regulating the ratio of Bcl-2/Bax expression［J］. Appl Physiol Nutr Metab, 2017, 42(8)：855-860.

［16］ Yan FX, Qian T, Gao WJ, et al. Effect of astragalus injection on the expression of Caspase-3 after hypoxia/hypoglycemia and reoxygenation in hippocampal neurons of rats［J］. Acta Anatomica Sinica,2011,42(1):14-21. (in Chinese)

闫凤霞, 钱涛, 高维娟,等. 黄芪注射液抑制缺氧缺糖/复氧复糖大鼠海马神经元Caspase-3表达［J］. 解剖学报, 2011, 42(1):14-21.