巨噬细胞迁移抑制因子对人胚胎干细胞存活、增殖和分化的影响  

Effects of macrophage migration inhibitory factor on survival,proliferation,and differentiation of human embryonic stem cells

作　　者：黄婷 郑晓晗 钟远吉 魏艳召 魏绪芳 曹旭东[4] 冯晓丽 赵振强[1,2,3] Huang Ting;Zheng Xiaohan;Zhong Yuanji;Wei Yanzhao;Wei Xufang;Cao Xudong;Feng Xiaoli;Zhao Zhenqiang(Department of Neurology,First Affiliated Hospital of Hainan Medical University,Haikou 570100,Hainan Province,China;Hainan Medical University,Haikou 570100,Hainan Province,China;Hainan Provincial Key Laboratory of Tropical Brain Research and Translation,Haikou 570100,Hainan Province,China;University of Ottawa,Ottawa K1N6N5,Ontario,Canada)

机构地区：[1]海南医学院第一附属医院神经内科,海南省海口市570100 [2]海南医学院,海南省海口市570100 [3]海南省热带脑科学研究与转化重点实验室,海南省海口市570100 [4]渥太华大学,加拿大渥太华K1N6N5

出　　处：《中国组织工程研究》2025年第7期1380-1387,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金(81860238),项目负责人:赵振强;海南省重点研发计划(ZDYF2018233),项目负责人:赵振强;海南省科技厅高层次人才项目(821RC694),项目负责人:赵振强;海南省国际科技合作人才与交流项目(G20241024014E),项目负责人:赵振强;海南省临床医学中心建设项目(琼卫医函[2021]276号);海南省卫生健康行业科研项目(21A200354),项目负责人:冯晓丽。

摘　　要：背景:巨噬细胞迁移抑制因子(macrophage migration inhibitory factor,MIF)是一种具有多效性作用的细胞因子,可以在不同类型干细胞中自分泌并且能调控细胞的增殖、分化和迁移。课题组前期研究证实人胚胎干细胞自分泌MIF,且在培养液中浓度基本固定。然而,MIF是否参与了人胚胎干细胞的存活、增殖和分化尚不清楚。目的:探究MIF对人胚胎干细胞存活、增殖和分化的作用。方法:(1)培养人胚胎干细胞H9,CCK-8法检测并绘制细胞生长曲线,采用酶联免疫吸附法定量检测培养基中MIF水平。(2)为了明确外源性MIF对人胚胎干细胞存活、增殖的影响,分为:对照组,细胞在干细胞培养基中正常培养;外源性MIF组,在干细胞培养基中分别添加30,100,300 ng/m L的MIF;MIF抑制剂ISO-1组,在干细胞培养基中分别添加2,7,21μmol/L的ISO-1;MIF+ISO-1组,在不同浓度ISO-1组中分别添加100 ng/m L MIF,采用CCK-8法检测上述各组细胞活力。(3)为进一步阐明MIF基因对人胚胎干细胞存活、增殖的影响,采用CRISPRCas9技术构建MIF敲除的H9细胞系,观察建系情况。(4)为了明确高浓度MIF对人胚胎干细胞初步分化是否有影响,在培养基中分别添加100 ng/m L MIF和100 ng/m L CXCR4中和抗体,采用实时荧光定量聚合酶链式反应(RT-q PCR)、免疫细胞荧光、蛋白质印迹法(Western blot)检测干细胞自我更新因子(KLF4、c-MYC、NANOG、OCT4、SOX2)及分化转录因子(FOXA2、OTX2)的表达水平。结果与结论:(1)人胚胎干细胞H9的对数生长期为3-6 d,正常生长的情况下自分泌MIF水平约为20 ng/m L,与细胞量无关;(2)与对照组相比,添加不同质量浓度MIF对人胚胎干细胞的增殖无影响(P>0.05);ISO-1明显抑制人胚胎干细胞的增殖,ISO-1浓度越大,抑制越明显(P<0.05);ISO-1中添加MIF可以减少ISO-1的抑制作用(P<0.05);(3)RT-q PCR检测MIF基因敲除约50%后,人胚胎干细胞生长活力显著降低并且无法建系成功;(4)�BACKGROUND:Macrophage migration inhibitory factor(MIF) is a pleiotropic cytokine,which is secreted in different types of stem cells and can regulate the proliferation,differentiation and migration of various types of stem cells.Our previous research has confirmed that human embryonic stem cells secrete MIF and that its concentration in the culture medium is relatively stable.However,whether MIF is involved in the survival,proliferation and differentiation of human embryonic stem cells remains unclear.OBJECTIVE:To investigate the effects of MIF on survival,proliferation,and differentiation of human embryonic stem cells.METHODS:(1) Human embryonic stem cells H9 were cultured.The growth curve of cells was detected and plotted by CCK-8 assay.Enzyme-linked immunosorbent assay was used to determine the level of MIF in the medium.(2) To determine the effects of exogenous MIF on the survival and proliferation of human embryonic stem cells,different groups were established:the control group,which was cultured in stem cell medium without any modifications;the exogenous MIF group,which was treated with different concentrations(30,100,300 ng/mL) of MIF in the stem cell medium;the MIF inhibitor ISO-1group,which was treated with different concentrations(2,7,21 μmol/L) of ISO-1 in the stem cell medium;and the MIF+ISO-1 group,which was treated with different concentrations of ISO-1 along with 100 ng/mL of MIF.Cell viability was assessed using the CCK-8 assay.(3) To further elucidate the effect of MIF gene on survival and proliferation of human embryonic stem cell,the MIF knockout H9 cell line was constructed by CRISPR-Cas 9 technology to observe the lineage establishment.(4) To determine the effect of high concentrations of MIF on human embryonic stem cell differentiation,100 ng/mL MIF and 100 ng/mL of CXCR4 neutralizing antibody were separately added to the normal stem cell culture medium.The expression levels of self-renewal factors(KLF4,c-MYC,NANOG,OCT4,and SOX2) and differentiation transcription factors(FOXA2,OTX2) were mea

关 键 词：巨噬细胞迁移抑制因子 人胚胎干细胞 自分泌 存活 分化 CXCR4 KLF4 FOXA2 

分 类 号：R459.9[医药卫生—治疗学] R318[医药卫生—临床医学] R394.2