二甲双胍(Metformin)在少突胶质前体细胞分化中的作用和机制<sup>*</sup>

doi:10.13523/j.cb.2103013

中国生物工程杂志

2021, Vol. 41

Issue (9): 1-9 DOI: 10.13523/j.cb.2103013

研究报告

二甲双胍(Metformin)在少突胶质前体细胞分化中的作用和机制^*

谈沛林^1,²,张莹²,张竣¹,高笑¹,王树坤²,侯琳¹,袁增强^1,^2,^**(

)

1 青岛大学基础医学院青岛 266071
2 军事科学院军事医学研究院军事认知与脑科学研究所北京 100850

Role and Mechanism of Metformin in Oligodendrocyte Precursor Cell Differentiation

TAN Pei-lin^1,²,ZHANG Ying²,ZHANG Jun¹,GAO Xiao¹,WANG Shu-kun²,HOU Lin¹,YUAN Zeng-qiang^1,^2,^**(

)

1 School of Basic Medicine, Qingdao 266071, China
2 Institute of Military Cognition and Brain Sciences, Academy of Military Medical Sciences, Beijing 100850, China

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摘要：

目的：研究二甲双胍(metformin)在少突胶质前体细胞(oligodendrocyte precursor cell, OPC)分化过程中的作用,并对其分子机制进行初步探讨。方法：使用免疫吸附法直接分离纯化OPC后诱导培养,通过免疫荧光染色对细胞进行鉴定。在不同浓度二甲双胍处理OPC后,使用CCK8检测细胞活性;通过免疫荧光染色、流式细胞分析、实时荧光定量PCR和蛋白质印迹检测二甲双胍对OPC分化中细胞数量、mRNA和蛋白质水平的影响。结果：使用免疫吸附法可分离出高纯度OPC;CCK8检测结果显示在100 μmol/L浓度以内,二甲双胍对细胞无毒性;免疫荧光染色结果显示,二甲双胍处理OPC后,PDGFRα ⁺ OLIG2⁺阳性细胞数明显增加,且MBP⁺细胞数显著增加;流式细胞分析结果显示,PDGFRα⁺细胞数显著增加;实时荧光定量PCR结果显示,OPC分化相关基因Mag、Mbp等的mRNA水平显著增加;蛋白质印迹结果显示,分化相关蛋白OLIG2和MBP表达增加。机制上,少突胶质细胞系Oli-neu、OPC分别经二甲双胍处理5 min后,RAS、p-MEK、p-ERK蛋白量显著增加。结论：二甲双胍通过RAS-MEK-ERK信号通路促进少突胶质前体细胞的分化。

关键词： 二甲双胍; 少突胶质前体细胞; 脱髓鞘疾病

Abstract:

Objective:To demonstrate the role of metformin in oligodendrocyte precursor cell (OPC) differentiation and preliminarily discuss the molecular mechanism.Methods:OPC was directly isolated and purified by immune adsorption from the brain and identified using immunofluorescence. Firstly, the concentration of metformin was decided through cell viability assay. Then, the effects of metformin on OPC-differentiation related positive cells, the mRNA or protein level were analyzed by immunofluorescence, flow cytometry, qRT-PCR, and western blot.Results:High purity of primary cells were obtained. CCK8 assay showed that there is no significant toxicity of metformin (<100 μmol/L) on cell viability. Moreover, the significant increasement of PDGFRα⁺OLIG2⁺ and MBP⁺ cells, up-regulation levels of Mag, Olig2, Mbp and Sox10 mRNA and OLIG2, MBP protein were detected in OPC after metformin treatment. Mechanically, compared to the control group, RAS, p-MEK and p-ERK proteins were significantly increased after metformin treatment for 5min in Oli-neu cells and OPC. Conclusion:Metformin promotes the differentiation of oligodendrocyte precursor cells through the RAS-MEK-ERK signaling pathway.

Key words: Metformin Oligodendrocyte precursor cells(OPCs) Demyelinating diseases

收稿日期: 2021-03-09 出版日期: 2021-09-30

ZTFLH:

Q819

基金资助: * 国家自然科学基金(81930029);国家自然科学基金(81971091);国家重点研发计划(2017YFA0106200);山东省重点研发计划(2019GSF107025)

通讯作者: 袁增强 E-mail: zyuan620@yahoo.com

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引用本文:

谈沛林,张莹,张竣,高笑,王树坤,侯琳,袁增强. 二甲双胍(Metformin)在少突胶质前体细胞分化中的作用和机制^*[J]. 中国生物工程杂志, 2021, 41(9): 1-9.

TAN Pei-lin,ZHANG Ying,ZHANG Jun,GAO Xiao,WANG Shu-kun,HOU Lin,YUAN Zeng-qiang. Role and Mechanism of Metformin in Oligodendrocyte Precursor Cell Differentiation. China Biotechnology, 2021, 41(9): 1-9.

链接本文:

https://manu60.magtech.com.cn/biotech/CN/10.13523/j.cb.2103013 或 https://manu60.magtech.com.cn/biotech/CN/Y2021/V41/I9/1

表1 qRT-PCR引物序列

图1 少突胶质前体细胞体外分离培养

图2 二甲双胍对细胞活力和蛋白表达的影响

图3 二甲双胍促进OPC分化

图4 二甲双胍促进OPC分化的机制

图5 二甲双胍调控少突胶质细胞分化

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