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J Zhejiang Univ (Med Sci)  2021, Vol. 50 Issue (5): 614-620    DOI: 10.3724/zdxbyxb-2021-0343
    
Establishment of a hypobaric hypoxia-induced cell injury model in PC12 cells
ZHANG Dongmei1,CAO Qilu1,JING Linlin1,ZHAO Xiuhua2,*(),MA Huiping1,*()
1. Key Laboratory of the Plateau Medicine, the 940th Hospital of Joint Logistics Support Force of Chinese People’s Liberation Army, Lanzhou 730050, China;
2. Second Department of Infectious Disease Control and Prevention, Center for Disease Control and Prevention, Western War Zone, Chengdu 610000, China
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Abstract  

Objective: To construct a hypobaric hypoxia-induced cell injury model. Methods:Rat pheochromocytoma PC12 cells were randomly divided into control group, normobaric hypoxia group and hypobaric hypoxia group. The cells in control group were cultured at normal condition, while cells in other two groups were cultured in normobaric hypoxia and hypobaric hypoxia conditions, respectively. CCK-8 method was used to detect cell viability to determine the optimal modeling conditions like the oxygen concentration, atmospheric pressure and low-pressure hypoxia time. The contents of lactate dehydrogenase (LDH), superoxide dismutase (SOD) and malondialdehyde (MDA) were detected by microplate method. The apoptosis ratio and cell cycle were analyzed by flow cytometry. Results: The hypobaric hypoxia-induced cell injury model can be established by culturing for 24?h at 1% oxygen concentration and 41?kPa atmospheric pressure. Compared with the control group and normobaric hypoxia group, the activity of LDH and the content of MDA in hypobaric hypoxia group were significantly increased, the activity of SOD was decreased, the percentage of apoptosis was increased (all P<0.05), and the cell cycle was arrested in G0/G1 phase.Conclusion: A stable and reliable cell injury model induced by hypobaric hypoxia has been established with PC12 cells, which provides a suitable cell model for the in vitro experimental study on nerve injury induced by hypoxia at high altitude.



Key wordsPC12 cells      Hypobaric hypoxia      Lactate dehydrogenase      Superoxide dismutase      Malondialdehyde      Apoptosis      Cell cycle     
Received: 20 February 2021      Published: 29 December 2021
CLC:  R966  
Corresponding Authors: ZHAO Xiuhua,MA Huiping     E-mail: zhaoxiuhua2021xz@163.com
Cite this article:

ZHANG Dongmei,CAO Qilu,JING Linlin,ZHAO Xiuhua,MA Huiping. Establishment of a hypobaric hypoxia-induced cell injury model in PC12 cells. J Zhejiang Univ (Med Sci), 2021, 50(5): 614-620.

URL:

https://www.zjujournals.com/med/10.3724/zdxbyxb-2021-0343     OR     https://www.zjujournals.com/med/Y2021/V50/I5/614


PC12细胞低压性缺氧损伤模型的建立

目的:构建PC12细胞低压性缺氧细胞模型。方法:将PC12细胞依据缺氧条件随机分为正常对照组、常压缺氧组、低压缺氧组;正常对照组以正常条件培养,常压缺氧组和低压缺氧组分别以常压缺氧和低压缺氧条件培养。通过考察氧气浓度、大气压力和低压缺氧时间,应用CCK-8法测定细胞活性,筛选低压缺氧的条件;显微镜下观察各组细胞形态、结构、数目,微板法检测细胞乳酸脱氢酶(LDH)、超氧化物歧化酶(SOD)活性和丙二醛(MDA)含量,流式细胞仪测定细胞凋亡比例和细胞周期。结果:在1%氧浓度和41?kPa低压缺氧环境下培养24h可建立低压性缺氧细胞模型。与正常对照组和常压缺氧组比较,低压缺氧组LDH活性及细胞MDA含量增加、SOD活性下降,细胞凋亡比例上升(均P<0.05),细胞周期阻滞发生在G0/G1期。结论:建立了稳定可靠的低压性缺氧损伤PC12细胞模型,可用于高原低压性缺氧神经损伤的体外实验研究。


关键词: PC12细胞,  低压性缺氧,  乳酸脱氢酶,  超氧化物歧化酶,  丙二醛,  细胞凋亡,  细胞周期 
Figure 1 Effects of different hypoxia concentrations on the activity of PC12 cells
Figure 2 Effects of atmospheric pressure on PC12 cell activity under 1% oxygen concentration
Figure 3 Effects of different culture time on PC12 cell activity under hypobaric hypoxia condition
Figure 4 Morphology of PC12 cells cultured under different conditions for 24 h

组别

n

乳酸脱氢酶(U/mL)

丙二醛(nmol/mg)

超氧化物歧化酶(U/mg)

正常对照组

6

22.55 ±2.11

0.0345±0.0011

0.123±0.101

常压缺氧组

6

38.15 ±1.99*

0.0683±0.0054*

0.116±0.083*

低压缺氧组

6

43.15 ±3.25**#

0.0789±0.0019**#

0.112±0.052**#

Table 1 Lactate dehydrogenase activity, malondialdehyde content and superoxide dismutase activity in each group
Figure 5 Results of apoptosis detected by flow cytometry
Figure 6 Cell cycles of PC12 cells in each group
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