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浙江大学学报（医学版）
浙江大学学报（医学版）  2021, Vol. 50 Issue (5): 614-620    DOI: 10.3724/zdxbyxb-2021-0343
原著     
PC12细胞低压性缺氧损伤模型的建立
张冬梅1,曹琪璐1,景临林1,赵秀华2,*(),马慧萍1,*()
1.中国人民解放军联勤保障部队第九四〇医院药剂科 全军高原医学实验室，甘肃兰州 730050
2.中国人民解放军西部战区疾病预防控制中心传染病防控二科，四川 成都 610000
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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摘要：

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    
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 words: PC12 cells    Hypobaric hypoxia    Lactate dehydrogenase    Superoxide dismutase    Malondialdehyde    Apoptosis    Cell cycle
收稿日期: 2021-02-20 出版日期: 2021-12-29
:  R966  
基金资助: 国家自然科学基金（81571847，81872796）； 军队后勤科研计划（CWH17J010，CLB18J028）
通讯作者: 赵秀华,马慧萍     E-mail: zhaoxiuhua2021xz@163.com
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引用本文:

张冬梅,曹琪璐,景临林,赵秀华,马慧萍. PC12细胞低压性缺氧损伤模型的建立[J]. 浙江大学学报（医学版）, 2021, 50(5): 614-620.

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.

链接本文:

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

图 1  不同氧浓度细胞活性比较与正常对照组比较，<0.01；与1%氧浓度比较，<0.01.
图 2  1%氧浓度下不同大气压力细胞活性比较与常压缺氧组比较，<0.05；与41 kPa压力比较，<0.05，<0.01；与47 kPa压力比较，<0.05，<0.01.
图 3  低压缺氧条件下不同培养时间点细胞活性比较与正常对照组比较，<0.01；与常压缺氧组比较，<0.01.
图 4  各组细胞培养24 h后光镜下形态A:正常对照组细胞贴壁生长状态良好，边缘清晰可见，多数伸出轴突（蓝色箭头所示），呈梭形状态（红色箭头所示）；B：常压缺氧组细胞形态固缩，伸出轴突的细胞数量减少（蓝色箭头所示），多数细胞成圆形（红色箭头所示），细胞膜较粗糙，细胞贴壁性较差；C：低压缺氧组细胞肿胀（绿色箭头所示），死细胞增多（橙色箭头所示），细胞生长停滞. 标尺=100 μm.

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**#
表 1  各组乳酸脱氢酶活性、丙二醛含量和超氧化物歧化酶活性比较
图 5  流式细胞术检测各组细胞凋亡结果
图 6  各组细胞周期检测结果及比较与正常对照组比较，<0.05.
1 ZHANGP, CHENJ S, LIQ Y, et al.Neuroprotectants attenuate hypobaric hypoxia-induced brain injuries in cynomolgus monkeys[J]Zool Res, 2021, 42( 1): 3-19.
doi: 10.24272/j.issn.2095-8137.2020.384
2 SHARMAP, TULSAWANIR. Ganoderma lucidum aqueous extract prevents hypobaric hypoxia induced memory deficit by modulating neurotransmission, neuroplasticity and maintaining redox homeostasis[J]Sci Rep, 2020, 10( 1): 8944.
doi: 10.1038/s41598-020-65812-5
3 陈建, 黄缄, 高钰琪, 等. 预缺氧对PC12细胞缺氧后细胞内游离Ca2+的影响[J]. 西北国防医学杂志, 2008, 29(4): 250-253
CHEN Jian, HUANG Jian, GAO Yuqi, et al. Effect of hypoxia preconditioning on intracellular Ca2+ of PC12 cells[J]. Medical Journal of National Defending Forces in Northwest China, 2008, 29(4): 250-253. (in Chinese)
4 闫向丽, 王利胜, 王圣鑫. HT22海马神经元细胞氧糖剥夺再灌注模型的建立[J]. 广州中医药大学学报, 2020, 37(181): 161-165
YAN Xiangli, WANG Lisheng, WANG Shengxin. Establishment of HT22 cell oxygen-glucose deprivation reperfusion model[J]. Journal of Guangdong Pharmaceutical University， 2020, 37(181): 161-165. (in Chinese)
5 刘菲, 张昊, 刘博, 等. 氯化钴诱导的N2a细胞缺氧损伤模型的机制研究[J]. 广东药科大学学报, 2020, 36(2): 249-253
LIU Fei, ZHANG Hao, LIU Bo, et al. Mechanism of cobalt chloride induced hypoxia on N2a cells[J]. Journal of Guangzhou University of Traditional Chinese Medicine, 2020, 36(2): 249-253. (in Chinese)
6 李海丽, 卢永昌, 钱帅, 等. 苞叶雪莲粗提物对EA. hy926细胞缺氧损伤的保护作用[J]. 湖北农业科学, 2016, 55(1): 167-169
LI Haili, LU Yongchang, QIAN Shuai, et al. Saussurea obvallata crude protective effects of hypoxia on EA. hy926 injury[J]. Hubei Agricultural Sciences, 2016, 55(1): 167-169. (in Chinese)
7 HEILJ, SCHULTZED, SCHEMMERP, et al.N-acetylcysteine protects hepatocytes from hypoxia-related cell injury[J]Clin Exp Hepatol, 2018, 4( 4): 260-266.
doi: 10.5114/ceh.2018.80128
8 冯岚, 张二龙, 高钰琪, 等. 7种不同组织来源细胞缺氧损伤效应评价[J]. 第三军医大学学报, 2019, 41(6): 570-574
FENG Lan, ZHANG Erlong, GAO Yuqi, et al. Evaluation of hypoxic injuries in 7 cell lines derived from different human tissues[J]. Journal of Third Military Medical University, 2019, 41(6): 570-574. (in Chinese)
9 牛其芳, 李德龙, 杨杨, 等. 人血管内皮细胞缺氧复氧损伤细胞模型的建立[J]. 中国口腔颌面外科杂志, 2019, 17(4): 295-299
NIU Qifang, LI Delong, YANG Yang, et al. Establishment of human vascular endothelial hypoxia/reoxygeneration injury cell model[J]. China Journal of Oral and Maxillofacial Surgery, 2019， 17(4): 295-299. (in Chinese)
10 刘阳, 安美文, 芦莹, 等. 压力下角质形成细胞与成纤维细胞共培养对细胞增殖及胶原合成的影响[J]. 医用生物力学, 2015, 30(2): 148-153
LIU Yang, AN Meiwen, LU Ying, et al. Effects of keratinocytes/fibroblasts co-culture under pressure on cell proliferation and collagen synthesis[J]. Journal of Medical Biomechanics, 2015, 30(2): 148-153. (in Chinese)
11 黄正, 刘益杰, 褚立希, 等. 周期性压力通过凋亡基因调节兔软骨细胞增殖和凋亡的实验研究[J]. 国际骨科学杂志, 2014, 35(3): 205-208
HUANG Zheng, LIU Yijie, CHU Lixi, et al. Periodic pressure regulates the proliferation and apoptosis of rabbit chondrocytes genes[J]. International Journal of Orthopaedics, 2014, 35(3): 205-208. (in Chinese)
12 COBELENSP M, VAN PUTTEB P, KAVELAARSA, et al.Inflammatory consequences of lung ischemia-reperfusion injury and low-pressure ventilation[J]J Surg Res, 2009, 153( 2): 295-301.
doi: 10.1016/j.jss.2008.04.022
13 TSENGV, NIK, ALLAWZIA, et al.Extracellular superoxide dismutase regulates early vascular hyaluronan remodeling in hypoxic pulmonary hypertension[J]Sci Rep, 2020, 10( 1): 280.
doi: 10.1038/s41598-019-57147-7
14 ASANOA, UEDAS, KUJII, et al.Intracellular hypoxia measured by 18F-fluoromisonidazole positron emission tomography has prognostic impact in patients with estrogen receptor-positive breast cancer[J]Breast Cancer Res, 2018, 20( 1): 78.
doi: 10.1186/s13058-018-0970-6
15 SHIQ, YUC, ZHUD, et al.Effects of dietary Sargassum horneri on resisting hypoxia stress, which changes blood biochemistry, antioxidant status, and hepatic HSP mRNA expressions of juvenile black sea bream Acanthopagrus Schlegelii[J]J Appl Phycol, 2020, 32( 5): 3457-3466.
doi: 10.1007/s10811-020-02132-1
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