解剖学报

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 • 上一篇    下一篇

 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*   

  1. 河北中医学院河北省心脑血管病中医药防治重点实验室，石家庄 050091
  • 收稿日期:2018-03-12 修回日期:2018-05-07 出版日期:2019-02-06 发布日期:2019-04-18
  • 通讯作者: 高维娟 E-mail:gwj6088@163.com
  • 基金资助:
    河北省应用基础研究计划重点项目;河北省2017年博士学位点科研条件建设项目;河北省普通高校高层次人才科学研究计划项目

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*   

  1. Heibei Key Laboratory of Chinese Medicine Research on CardioCerebrovascular Disease, Hebei University of Chinese Medicine，Shijiazhuang 050091, China
  • Received:2018-03-12 Revised:2018-05-07 Online:2019-02-06 Published:2019-04-18
  • Contact: GAO Wei-juan E-mail:gwj6088@163.com

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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。

关键词: 缺氧缺糖/复氧复糖, 细胞损伤, 动态观察, 免疫荧光, HT22细胞

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.

Key words: Oxygen and glucose deprivation/reoxygenation, Cell injury, Dynamic observation, Immunofluorescence, HT22 cell