摘要
目的探究白细胞介素1β(IL-1β)调控人脐带间充质干细胞CD200表达及其对巨噬细胞极化的影响及作用机制。方法无血清培养基分离培养获得人脐带间充质干细胞(hUC-MSC),形态学观察及流式细胞术检测CD73、CD90、CD105、CD14、CD34、CD45、人类白细胞抗原DR(HLA-DR)的表达,确定间充质干细胞属性;20 ng/mL IL-1β处理hUC-MSC 24 h,流式细胞术检测CD200阳性细胞率,实时定量PCR和Western blot法检测CD200 mRNA和蛋白表达水平;佛波酯(PMA)诱导THP-1巨噬细胞活化,并与IL-1β处理感染CD200过表达慢病毒的hUC-MSC共培养,流式细胞术检测CD11c和CD206阳性细胞比例;IL-1β联合细胞外信号调节激酶1/2(ERK1/2)特异性抑制剂PD98059处理hUC-MSC,Western blot法检测细胞丝裂原激活蛋白激酶(MAPK)信号分子与CD200的表达。结果IL-1β显著下调hUC-MSC CD200蛋白表达与CD200阳性细胞率;过表达CD200显著上调hUC-MSC CD200表达,且CD200过表达hUC-MSC提高巨噬细胞CD206阳性细胞比率;IL-1β激活hUC-MSC的ERK1/2信号通路,PD98059上调IL-1β处理后hUC-MSC中CD200的蛋白表达。结论IL-1β通过激活ERK1/2信号通路抑制CD200的表达,进而抑制hUC-MSC对巨噬细胞向M2型极化的促进作用。
Objective To investigate the regulation of IL-1βon the expression of CD200 in human umbilical cord mesenchymal stem cells(hUC-MSCs),its role in macrophage polarization and the underlying mechanism.Methods hUC-MSCs were isolated and cultured in serum-free medium.Morphological observation and the expressions of CD73,CD90,CD105,CD14,CD34,CD45 and HLA-DR were detected by flow cytometry to confirm the properties of mesenchymal stem cells.hUC-MSCs were treated with IL-1βat the final concentration of 20 ng/mL for 24 hours.The proportion of CD200 positive cells was measured by flow cytometry.Real-time quantitative PCR and Western blot analysis were used to detect CD200 mRNA and protein expression levels.hUC-MSCs infected with CD200 overexpression(OE-CD200)and its negative control(OE-NC)lectin virus were treated with IL-1βand co-cultured with PMA-activated THP-1 macrophages.The proportion of CD11c and CD206 positive cells was measured by flow cytometry.hUC-MSCs were treated with IL-1βin combination with PD98059,and the expression of MAPK signaling pathway-related proteins and its effect on CD200 expression were detected by Western blot analysis.Results IL-1βsignificantly down-regulated the expression of CD200 protein and the proportion of CD200 positive cells.Overexpression of CD200 significantly up-regulated the expression of CD200 in hUC-MSCs,and increased the proportion of CD206-positive macrophages.IL-1βactivated the ERK1/2 signaling pathway in hUC-MSCs,and PD98059 up-regulated the expression of CD200 protein in hUC-MSCs treated with IL-1β.Conclusion IL-1βinhibits the expression of CD200 by activating ERK1/2 signaling pathway,and reduces the immunosuppressive effect of hUC-MSCs on regulating the M2-type polarization of macrophages.
作者
朱永朝
李莉
王拯
谭希鹏
陶金
丁璐
董辉
叶鹏
ZHU Yongzhao;LI Li;WANG Zheng;TAN Xipeng;TAO Jin;Ding Lu;DONG Hui;YE Peng(Institute of Medical Sciences,General Hospital of Ningxia Medical University,Yinchuan 750004,China)
出处
《细胞与分子免疫学杂志》
CAS
CSCD
北大核心
2024年第3期193-198,共6页
Chinese Journal of Cellular and Molecular Immunology
基金
国家自然科学基金(82060020)
宁夏回族自治区重点研发项目(2022BEG03088,2021BEG02032)
宁夏自然科学基金(2020AAC03387)。
