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ENGLISH ABSTRACT
M2型小胶质细胞源性外泌体与小鼠神经元氧糖剥夺/复糖复氧损伤的关系
单凯悦
李慧敏
王红
陈静燕
刘文洁
陈怀龙
张高峰
王明山
董瑞
作者及单位信息
·
DOI： 10.3760/cma.j.cn131073.20220413.01016
Relationship between M2-type microglia-derived exosomes and neuronal oxygen-glucose deprivation and restoration injury in mice
Shan Kaiyue
Li Huimin
Wang Hong
Chen Jingyan
Liu Wenjie
Chen Huailong
Zhang Gaofeng
Wang Mingshan
Dong Rui
Authors Info & Affiliations
Shan Kaiyue
Graduate School of Dalian Medical University, Dalian 116044, China
Department of Anesthesiology, Qingdao Municipal Hospital Affiliated to Qingdao University, Qingdao 266071, China
Li Huimin
Department of Anesthesiology, Qingdao Municipal Hospital Affiliated to Qingdao University, Qingdao 266071, China
Wang Hong
Department of Education and Training, Qingdao Women and Children′s Hospital, Qingdao University, Qingdao 266071, China
Chen Jingyan
Department of Anesthesiology, Qingdao Medical College of Nanjing Medical University, Qingdao 266071, China
Liu Wenjie
Department of Anesthesiology, Qingdao Municipal Hospital Affiliated to Qingdao University, Qingdao 266071, China
Chen Huailong
Department of Anesthesiology, Qingdao Municipal Hospital Affiliated to Qingdao University, Qingdao 266071, China
Zhang Gaofeng
Department of Anesthesiology, Qingdao Municipal Hospital Affiliated to Qingdao University, Qingdao 266071, China
Wang Mingshan
Department of Anesthesiology, Qingdao Municipal Hospital Affiliated to Qingdao University, Qingdao 266071, China
Dong Rui
Department of Anesthesiology, Qingdao Municipal Hospital Affiliated to Qingdao University, Qingdao 266071, China
Medical School, Nanjing University, Nanjing 210093, China
·
DOI： 10.3760/cma.j.cn131073.20220413.01016
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摘要

目的评价M2型小胶质细胞源性外泌体(M2-exo)与小鼠神经元氧糖剥夺/复糖复氧(OGD/R)损伤的关系。

方法体外培养小鼠神经母细胞(N2a细胞)及BV2小胶质细胞，采用20 ng/ml IL-4将BV2小胶质细胞激活为M2型，提取M0型小胶质细胞源性外泌体(M0-exo)和M2-exo。采用随机数字表法将N2a细胞分为4组( n＝23)：对照+M0-exo组(C+M0组)、对照+M2-exo组(C+M2组)、OGD/R+M0-exo组(O+M0组)和OGD/R+M2-exo组(O+M2组)。C+M0组和C+M2组分别加入M0-exo和M2-exo(终浓度100 μg/ml)孵育24 h，O+M0组和O+M2组于氧糖剥夺3 h时分别加入M0-exo和M2-exo(终浓度100 μg/ml)，再复糖复氧24 h。采用CCK-8法检测N2a细胞活力，采用LDH释放率评估细胞损伤程度，采用Western blot法及实时q-PCR检测Bax和Bcl-2及其mRNA表达。

结果与C+M0组比较，C+M2组N2a细胞活力、LDH释放率、Bax/Bcl-2比值和Bax mRNA/Bcl-2 mRNA比值差异无统计学意义( P>0.05)，O+M0组N2a细胞活力下降，LDH释放率、Bax/Bcl-2比值和Bax mRNA/Bcl-2 mRNA比值升高( P<0.05)。与C+M2组比较，O+M2组N2a细胞活力下降，LDH释放率、Bax/Bcl-2比值和Bax mRNA/Bcl-2 mRNA比值升高( P<0.05)。与O+M0组比较，O+M2组N2a细胞活力升高，LDH释放率、Bax/Bcl-2比值和Bax mRNA/Bcl-2 mRNA比值降低( P<0.05)。

结论M2-exo在小鼠神经元OGD/R过程中发挥内源性保护作用，可能与抑制细胞凋亡有关。

小神经胶质细胞;外泌体;神经元;再灌注损伤
ABSTRACT

ObjectiveTo evaluate the relationship between M2-type microglia-derived exosomes (M2-exo) and neuronal oxygen-glucose deprivation and restoration (OGD/R) injury in mice.

MethodsMouse neuroblastoma cells (N2a cells) and BV2 microglia were cultured in vitro, and BV2 microglia were activated to M2 type using 20 ng/ml IL-4, and M0-type microglia-derived exosomes (M0-exo) and M2-exo were extracted.N2a cells were divided into 4 groups ( n=23 each) using the random number table method: control+ M0-exo group (C+ M0 group), control+ M2-exo group (C+ M2 group), OGD/R+ M0-exo group (O+ M0 group) and OGD/R+ M2-exo group (O+ M2 group).M0-exo and M2-exo (final concentration 100 μg/ml) were added in C+ M0 and C+ M2 groups, respectively, and the cells were incubated for 24 h. M0-exo and M2-exo (final concentration 100 μg/ml) were added at 3 h after oxygen and glucose deprivation, and then the cells were incubated for 24 h in O+ M0 and O+ M2 groups, respectively.N2a cell viability was measured by the CCK-8 method, and the severity of cell damage was assessed using the lactic dehydrogenase (LDH) release rate.The expression of Bax and Bcl-2 protein and mRNA was detected by quantitative real-time polymerase chain reaction and Western blot.

ResultsCompared with C+ M0 group, no significant changes were found in N2a cell viability, LDH release rate, Bax/Bcl-2 ratio and Bax mRNA/Bcl-2 mRNA ratio in C+ M2 group ( P>0.05), and N2a cell viability was significantly decreased, and the LDH release rate, Bax/Bcl-2 ratio and Bax mRNA/Bcl-2 mRNA ratio were increased in O+ M0 group ( P<0.05).Compared with C+ M2 group, the N2a cell viability was significantly decreased, and the LDH release rate, Bax/Bcl-2 ratio and Bax mRNA/Bcl-2 mRNA ratio were increased in O+ M2 group ( P<0.05).Compared with O+ M0 group, N2a cell viability was significantly increased, and LDH release rate, Bax/Bcl-2 ratio, and Bax mRNA/Bcl-2 mRNA ratio were decreased in O+ M2 group ( P<0.05).

ConclusionsM2-exo exerts an endogenous protective effect during OGD/R in mouse neurons, which may be related to the inhibition of cell apoptosis.

Microglia;Exosomes;Neurons;Reperfusion injury
Dong Rui, Email: nc.defudabe.uhwiurgnod
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引用本文

单凯悦,李慧敏,王红,等. M2型小胶质细胞源性外泌体与小鼠神经元氧糖剥夺/复糖复氧损伤的关系[J]. 中华麻醉学杂志,2022,42(10)：1233-1237.

DOI:10.3760/cma.j.cn131073.20220413.01016

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脑缺血再灌注损伤的机制复杂，包括炎症、氧化应激、细胞凋亡、自噬等 [ 1 , 2 ]。小胶质细胞作为中枢神经系统免疫反应的主要效应细胞，通过对脑组织的永久监视来维持中枢神经系统稳态 [ 3 ]。当脑组织创伤、感染、缺血缺氧时，小胶质细胞迅速激活为高表达促炎因子的M1型或产生抗炎因子的M2型 [ 4 ]。脑缺血再灌注时，小胶质细胞激活参与神经系统炎症的发生与转归，其中M2型小胶质细胞以多种形式参与脑组织修复 [ 5 ]。外泌体是一种包含大量遗传物质的细胞外囊泡，可被周围细胞吸收或通过血液和淋巴液进入循环，最终作用于靶细胞发挥其生物学作用 [ 6 ]。外泌体具有强大的信息传递功能，可以作为功能性小RNA和蛋白质的天然载体进行疾病治疗。此外，外泌体可透过血脑屏障，有可能成为治疗中枢系统疾病的方法 [ 7 ]。研究表明，外伤性脑损伤后小胶质细胞及其外泌体中miR-124-3p表达上调，不仅抑制神经元的炎症反应，M2型小胶质细胞源性外泌体(M2-exo)还可转移到神经元中促进突触生长 [ 8 ]。M2-exo是否参神经元氧糖剥夺/复糖复氧(OGD/R)损伤的修复尚有待探讨。本研究拟评价M2-exo与小鼠神经元OGD/R损伤的关系，进一步探讨脑缺血再灌注损伤的机制。
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备注信息
A
董瑞，Email： nc.defudabe.uhwiurgnod
B

单凯悦、李慧敏：课题实施、论文撰写；王红、陈静燕、刘文洁：数据整理、统计学分析；陈怀龙、张高峰、王明山、董瑞：研究指导、论文修改、经费支持

C
所有作者声明无利益冲突
D
国家自然科学基金 (82001132)
贝朗麻醉科研基金 (BBDF-2019-010)
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