低氧暴露对小鼠脾淋巴细胞增殖和凋亡的影响

doi:10.11958/20220649

天津医药 ›› 2022, Vol. 50 ›› Issue (10): 1014-1019.doi: 10.11958/20220649

低氧暴露对小鼠脾淋巴细胞增殖和凋亡的影响

郭玉静(), 胡英, 龙启福, 许玉珍, 李积东, 永胜(),^△

青海大学医学院基础医学部免疫学教研室（邮编810016）

收稿日期:2022-04-27 修回日期:2022-06-07 出版日期:2022-10-15 发布日期:2022-10-20
通讯作者: ^△永胜 E-mail:ly47757@163.com;yongsheng@qhu.edu.cn
作者简介:郭玉静（1998）,女,硕士在读,主要从事低氧对免疫功能改变及适应机制方面研究。E-mail： ly47757@163.com
基金资助:
国家自然科学基金资助项目(82060295);青海省科技计划项目基金(2019-ZJ-7059)

Effects of hypoxia exposure on proliferation and apoptosis of mouse spleen lymphocytes

GUO Yujing(), HU Ying, LONG Qifu, XU Yuzhen, LI Jidong, YONG Sheng(),^△

Department of Immunology, School of Basic Medicine, Qinghai University School of Medical, Xining 810016, China

Received:2022-04-27 Revised:2022-06-07 Published:2022-10-15 Online:2022-10-20
Contact: ^△YONG Sheng E-mail:ly47757@163.com;yongsheng@qhu.edu.cn

郭玉静, 胡英, 龙启福, 许玉珍, 李积东, 永胜. 低氧暴露对小鼠脾淋巴细胞增殖和凋亡的影响[J]. 天津医药, 2022, 50(10): 1014-1019.

GUO Yujing, HU Ying, LONG Qifu, XU Yuzhen, LI Jidong, YONG Sheng. Effects of hypoxia exposure on proliferation and apoptosis of mouse spleen lymphocytes[J]. Tianjin Medical Journal, 2022, 50(10): 1014-1019.

摘要/Abstract

摘要：

目的研究低氧暴露诱导淋巴细胞数量减少与细胞增殖和凋亡的关系。方法从C57BL/6小鼠脾中分离淋巴细胞,将淋巴细胞分别在低氧（1%O₂）和常氧（21%O₂）环境中培养12、24、48 h。用流式细胞术检测T、B淋巴细胞数量;羧基荧光素乙酰乙酸和AnnexinⅤ-FITC/PI方法分别检测淋巴细胞的增殖和凋亡;扫描电镜观察淋巴细胞形态;实时荧光定量逆转录PCR和Western blot检测凋亡相关因子鼠源B细胞淋巴瘤/白血病-2（bcl-2）、bcl-2同源拮抗剂-杀伤蛋白（Bak）和胱天蛋白酶-3（caspase-3）的mRNA和蛋白表达水平。结果低氧暴露12、24、48h,可降低T、B淋巴细胞数量,抑制淋巴细胞增殖,促进细胞凋亡;扫描电镜观察显示,同一时间下,低氧组淋巴细胞更早出现凋亡特征形态学变化;低氧暴露12、24、48 h,淋巴细胞Bak和caspase-3的mRNA和蛋白表达水平均上调,而bcl-2的mRNA和蛋白表达水平均下调。结论低氧暴露通过抑制淋巴细胞增殖、促进凋亡,从而介导淋巴细胞数量下降。

关键词: 缺氧, 淋巴细胞减少, 细胞增殖, 细胞凋亡, bcl-2同源对抗杀伤蛋白, 胱天蛋白酶-3

Abstract:

Objective To investigate the relationship between lymphocyte number reduction, cell proliferation and apoptosis induced by hypoxia exposure. Methods Lymphocytes were isolated from spleen of C57BL/6 mice and cultured in hypoxia (1%O₂) and normoxia (21%O₂) for 12, 24 and 48 h, respectively. T and B lymphocytes were assessed by flow cytometry. Proliferation and apoptosis of lymphocytes were detected by CFSE and AnnexinⅤ-FITC/PI method, respectively. The morphology of lymphocytes was observed by scanning electron microscope. The mRNA and protein expression levels of apoptosis-related factor bcl-2 homologous antagonist killer protein (Bak), murine B-cell lymphoma/leukemia-2 (bcl-2) and cysteine aspartate protease-3 (caspase-3) were detected by real-time quantitative reverse transcription PCR (qPCR) and Western blot assay. Results Hypoxic exposure for 12, 24 and 48 h can reduce the number of T and B lymphocytes, inhibit the proliferation of lymphocytes and promote cell apoptosis. Scanning electron microscopy showed that at the same time, the characteristic morphological changes of apoptosis appeared earlier in the hypoxic group. After 12, 24 and 48 h hypoxia exposure, the mRNA and protein expression levels of Bak and caspase-3 in lymphocytes were up-regulated, while the mRNA and protein expression levels of bcl-2 were down-regulated. Conclusion Hypoxic exposure mediates the decline of lymphocyte numbers by inhibiting lymphocyte proliferation and promoting apoptosis.

Key words: hypoxia, lymphopenia, cell proliferation, apoptosis, bcl-2 homologous antagonist-killer protein, caspase 3

中图分类号:

R392.12

图/表 10

表1 qPCR引物序列

Tab. 1 Sequence of the primers in qPCR

基因名称	引物序列（5′→3′）	产物大小（bp）
β-actin	上游：CATCCGTAAAGACCTCTATGCCAAC	171
β-actin	下游：ATGGAGCCACCGATCCACA	171
Bak	上游：GGTCTTTCGAAGCTACGTTTTT	243
Bak	下游：ATCTTGGTGAAGAGTTCGTAGG	243
bcl-2	上游：GATGACTTCTCTCGTCGCTAC	156
bcl-2	下游：GAACTCAAAGAAGGCCACAATC	156
caspase-3	上游：GGCCTGAAATACCAAGTCAGGAA	128
caspase-3	下游：CCATGGCTTAGAATCACACACACA	128

表2 2组不同时间T、B淋巴细胞百分比比较（n=3,%,$\bar{x} \pm s$）

Tab. 2 Comparison of the percentages of T and B lymphocytes at different time points between the two groups

组别	12 h		24 h		48 h
组别	T淋巴细胞	B淋巴细胞	T淋巴细胞	B淋巴细胞	T淋巴细胞	B淋巴细胞
常氧组	40.17±1.90	33.47±2.38	32.65±4.60	27.96±1.50	26.30±1.11	24.62±2.88
低氧组	32.26±3.30	27.42±2.73	23.77±2.61	23.39±2.31	23.16±1.33	18.71±1.31
t	3.603^＊	2.895^＊	2.904^＊	2.873^＊	3.140^＊	3.229^＊

图1 常氧和低氧暴露不同时间对淋巴细胞增殖的影响

Fig.1 Effects of normoxia and hypoxia exposure on lymphocyte proliferation at different time points

表3 2组不同时间淋巴细胞增殖率比较（n=3,%,$\bar{x} \pm s$）

Tab. 3 Comparison of lymphocyte proliferation rates at different time points between two groups

组别	12 h	24 h	48 h
常氧组	31.56±1.63	38.50±0.31	43.32±1.35
低氧组	27.31±2.05	34.74±0.82	40.66±0.49
t	4.749^＊＊	11.324^＊＊	5.566^＊＊

表4 2组不同时间淋巴细胞凋亡率比较（n=3,%,$\bar{x} \pm s$）

Tab. 4 Comparison of apoptosis rate of lymphocyte at different time points between the two groups

组别	12 h	24 h	48 h
常氧组	17.05±1.33	30.66±3.11	82.01±1.66
低氧组	22.28±1.13	36.34±1.64	86.99±1.42
t	5.446^＊＊	3.172^＊	6.033^＊＊

图2 常氧和低氧暴露不同时间对淋巴细胞凋亡的影响

Fig.2 Effects of normoxia and hypoxia exposure on lymphocyte apoptosis at different time points

图3 SEM观察低氧暴露不同时间淋巴细胞形态变化

Fig.3 Lymphocyte morphological changes of hypoxia exposure at different time points observed by SEM

表5 2组低氧暴露不同时间淋巴细胞Bak、bcl-2、caspase-3 mRNA表达水平比较（n=3,$\bar{x} \pm s$）

Tab. 5 Comparison of mRNA expression levels of Bak, bcl-2 and caspase-3 in lymphocytes at different time points between the two groups

组别	12 h			24 h			48 h
组别	Bak	bcl-2	caspase-3	Bak	bcl-2	caspase-3	Bak	bcl-2	caspase-3
常氧组	0.98±0.12	1.00±0.10	0.96±0.08	1.01±0.36	1.10±0.30	0.94±0.08	0.95±0.49	1.16±0.47	0.98±0.16
低氧组	1.11±0.18	0.66±0.21	1.25±0.35	1.43±0.28	0.52±0.22	1.15±0.19	1.44±0.42	0.74±0.39	2.21±0.56
t	2.248^＊	4.527^＊＊	2.171^＊	2.724^＊	4.440^＊＊	2.532^＊	2.721^＊	2.516^＊	5.973^＊＊

表6 2组低氧暴露不同时间淋巴细胞Bak、bcl-2、caspase-3蛋白表达水平比较（n=3,$\bar{x} \pm s$）

Tab. 6 Comparison of Bak, bcl-2 and caspase-3 protein expression levels in lymphocytes at different time points between the two groups of mice

组别	12 h			24 h			48 h
组别	Bak	bcl-2	caspase-3	Bak	bcl-2	caspase-3	Bak	bcl-2	caspase-3
常氧组	0.31±0.11	0.62±0.06	0.45±0.08	0.23±0.15	0.90±0.14	0.71±0.06	0.37±0.13	0.36±0.04	0.36±0.03
低氧组	0.54±0.09	0.44±0.09	0.72±0.15	0.48±0.03	0.50±0.17	0.94±0.13	0.73±0.17	0.23±0.02	0.66±0.10
t	2.828^＊	2.819^＊	2.794^＊	2.799^＊	3.117^＊	2.787^＊	2.867^＊	4.658^＊＊	4.950^＊＊

图4 低氧暴露不同时间淋巴细胞Bak、bcl-2、caspase-3的蛋白表达水平

Fig.4 Protein expression levels of Bak, bcl-2 and caspase-3 in lymphocytes at different time points between the two groups of mice

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低氧暴露对小鼠脾淋巴细胞增殖和凋亡的影响

Effects of hypoxia exposure on proliferation and apoptosis of mouse spleen lymphocytes

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