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中华神经创伤外科电子杂志

中华神经创伤外科电子杂志 ›› 2023, Vol. 09 ›› Issue (02) : 69 -75. doi: 10.3877/cma.j.issn.2095-9141.2023.02.002

基础研究

三种模拟创伤性脑损伤体外细胞模型的损伤特征比较
阿迪莱·阿卜杜热西提, 费奥, 邢晓雯, 谢胜强, 张睿, 兰晓娟, 程岗†()   
  1. 510006　广州，华南理工大学医学院（生命科学研究院）
    100853　北京，解放军总医院京南医疗区门诊部
    100850　北京，军事科学院军事医学研究院脑科学中心
    100853　北京，解放军总医院第一医学中心神经外科医学部
    230031　合肥，安徽医科大学海军临床学院
  • 收稿日期:2023-01-24 出版日期:2023-04-15
  • 通信作者: 程岗

Comparison of injury features of three cell models simulating traumatic brain injury in vitro

Abudurxiti Adilai·, Ao Fei, Xiaowen Xing, Shengqiang Xie, Rui Zhang, Xiaojuan Lan, Gang Cheng†()   

  1. South China University of Technology School of Medicine (School of Life Sciences), Guangzhou 510006, China
    Department of Outpatient, Jingnan Medical District, General Hospital of the People's Liberation Army, Beijing 100853, China
    Brain Science Center, Academy of Military Medicine, Military Science, Beijing 100850, China
    Neurosurgery Institute, The First Medical Center, General Hospital of PLA, Beijing 100853, China
    Naval Clinical College of Anhui Medical University, Hefei 230031, China
  • Received:2023-01-24 Published:2023-04-15
  • Corresponding author: Gang Cheng
  • Supported by:
    PLA Medical Youth Support Project(17QNP014); PLA Equipment Project(LB20191A010010); PLA Equipment Project(LB20201A010026)
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引用本文：

阿迪莱·阿卜杜热西提, 费奥, 邢晓雯, 谢胜强, 张睿, 兰晓娟, 程岗. 三种模拟创伤性脑损伤体外细胞模型的损伤特征比较[J]. 中华神经创伤外科电子杂志, 2023, 09(02): 69-75.

Abudurxiti Adilai·, Ao Fei, Xiaowen Xing, Shengqiang Xie, Rui Zhang, Xiaojuan Lan, Gang Cheng. Comparison of injury features of three cell models simulating traumatic brain injury in vitro[J]. Chinese Journal of Neurotraumatic Surgery(Electronic Edition), 2023, 09(02): 69-75.

目的

系统比较拉伸模型、氧-糖剥夺（OGD）模型以及过氧化氢（H2O2）处理的氧化应激模型的细胞损伤特征，为TBI研究中细胞模型的选择提供参考。

方法

复苏冻存的HT-22细胞后，将HT-22细胞接种于六孔板中，分为实验组和对照组，实验组分别制备细胞拉伸损伤模型、OGD模型、氧化应激损伤模型，对照组正常培养，不做特殊处理。通过显微镜观察细胞形态的变化，CCK-8实验分析测试细胞活力；提取细胞蛋白，采用Western blot检测凋亡（Bcl-2、Bax）、坏死性凋亡（RIPK3、RIPK1）、铁死亡（ACSL4、GPX4、SLC7A11）等不同细胞死亡途径的标志物变化。

结果

与对照组相比，实验组的3种损伤模型均导致HT-22细胞出现不同程度的形态异常、增殖受限或脱落现象。CCK-8检测结果显示，不同模型处理后细胞存活率均较对照组明显降低，差异具有统计学意义（P<0.05）。Western blot检测结果显示，3.0 PSI强度的拉伸处理后细胞中的GPX4、SLC7A11、Bax、RIPK3、RIPK1蛋白表达较对照组明显增高，差异均有统计学意义（P<0.05）；OGD处理12 h后，SLC7A11和Bax表达较对照组明显上调，差异均有统计学意义（P<0.05）；800 μmol/L的H2O2处理后，Bcl-2和RIPK1蛋白较对照组表达明显上调，差异均有统计学意义（P<0.05）。

结论

拉伸损伤模型更能反映TBI后复杂的损伤特点，OGD与H2O2处理模型更适合用于研究特定的损伤通路。

关键词: 创伤性脑损伤, 拉伸损伤, 氧-糖剥夺, 氧化应激, 凋亡, 坏死性凋亡, 铁死亡
Objective

To systematically compare the cell damage characteristics of stretch injury model, oxygen-glucose deprivation (OGD) model and hydrogen peroxide (H2O2)-treated oxidative stress model, and to provide a reference for the selection of cell models in TBI research.

Methods

After resuscitation of frozen HT-22 cells, HT-22 cells were seeded in Six-well plates, which were divided into experimental group and control group. The experimental group prepared cell stretch injury models, OGD models, and oxidative stress injury models respectively. The control group was cultured normally without any special treatment. The changes of cell morphology were observed by microscope, and cell viability was tested by CCK-8 analysis; cell proteins were extracted, and changes of apoptosis markers (Bcl-2, Bax), necroptosis markers (RIPK3, RIPK1), ferroptosis markers (ACSL4, GPX4, SLC7A11) were detected by Western blot.

Results

Compared with the control group, all three injury models led to abnormal morphological changes in different degrees, restricted proliferation or cell shedding in HT-22 cells, indicating cell damage. CCK-8 analysis showed that the cell viability in different model treatment groups was significantly reduced (P<0.05). The results of Western blot demonstrated that the expression of GPX4, SLC7A11, Bax, RIPK3 and RIPK1 proteins in cells treated with 3.0 PSI tensile strength was significantly increased, with statistical significance (P<0.05). After OGD treatment for 12 h, the expression of SLC7A11 and Bax was significantly up-regulated, with statistical significance (P<0.05). After 800 μmol/L hydrogen peroxide treatment, the expression of Bcl-2 and RIPK1 proteins was significantly up-regulated, with statistical significance (P<0.05).

Conclusion

The stretch model can better reflect the complex injury characteristics after TBI, and the OGD and H2O2 treatment models are more suitable for studying specific injury pathways.

Key words: Traumatic brain injury, Stretch injury, Oxygen-glucose deprivation, Oxidative stretch injury, Apoptosis, Necroptosis, Ferroptosis
图1 不同处理组HT-22细胞形态变化A~F：实验组；A：拉伸损伤峰值压力为2.5 PSI（100×）；B：拉伸损伤峰值压力为3.0 PSI（100×）；C：OGD处理3 h（40×）；D：OGD处理12 h（40×）；E：200 μmol/L H2O2处理（40×）；F：800 μmol/L H2O2处理（40×）；G：对照组（100×）
Fig.1 Morphological changes of HT-22 cells in different models
图2 不同处理组细胞存活率比较与对照组比较，aP<0.05，n=3
Fig.2 Comparison of cell survival rates among different models
图3 细胞拉伸损伤模型中铁死亡、凋亡和坏死性凋亡相关蛋白表达变化A：蛋白Western blot条带图；B：相关蛋白表达水平；与对照组比较，aP<0.05，n=3
Fig.3 Expression of ferroptosis, apoptosis and necroptosis related marker proteins in cell stretch injury model
图4 OGD模型中铁死亡、凋亡和坏死性凋亡相关蛋白表达变化A：蛋白Western blot条带图；B：相关蛋白表达水平；与对照组比较，aP<0.05，n=3
Fig.4 Expression of ferroptosis，apoptosis and necroptosis related marker proteins in OGD model
图5 氧化应激损伤模型中铁死亡、凋亡和坏死性凋亡相关蛋白表达变化A：蛋白Western blot条带图；B：相关蛋白表达水平；与对照组比较，aP<0.05，n=3
Fig.5 Expression of ferroptosis，apoptosis and necroptosis related marker proteins in oxidative stress injury model
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