人脐血间充质干细胞分泌生物活性因子肿瘤坏死因子α-刺激基因-6蛋白对小鼠骨髓来源巨噬细胞亚型转化的影响  

Effects of human umbilical cord blood mesenchymal stem cells secreting tumor necrosis factor alpha stimulating gene-6 protein on conversion of mouse bone marrow-derived macrophage subtypes

作　　者：赵吉玲 彭漪[1] 彭智勇 余国龙[1] Zhao Jiling;Peng Yi;Peng Zhiyong;Yu Guolong(Department of Cardiology,Xiangya Hospital,Central South University,Changsha 410008,Hunan Province,China)

机构地区：[1]中南大学湘雅医院心内科,湖南省长沙市410008

出　　处：《中国组织工程研究》2022年第13期2012-2019,共8页Chinese Journal of Tissue Engineering Research

基　　金：国家自然科学基金面上项目(81570266),项目负责人:余国龙;湖南省自然科学基金重点项目(2015SK2025),项目负责人:余国龙。

摘　　要：背景:干细胞以旁分泌或远距分泌的方式对炎症反应产生调节作用,是干细胞移植治疗疾病的主要机制之一。移植的干细胞分泌大量生物活性分子,如转化生长因子β、前列腺素E2、白细胞介素10和肿瘤坏死因子α-刺激基因-6蛋白(tumor necrosis factorαstimulating gene-6 protein,TSG-6)等,影响单核/巨噬细胞从促炎M1表型转化为抑炎M2表型,协调促炎和抗炎因子的平衡,是其调节炎症反应的重要机制,而目前干细胞分泌因子促进单核/巨噬细胞亚型转化的作用机制尚不明确。目的:探讨人脐血间充质干细胞分泌的生物活性分子之一TSG-6对小鼠骨髓来源巨噬细胞表型变化及炎症因子水平的影响,并探究TSG-6蛋白介导的巨噬细胞M2极化的机制。方法:①无菌提取6-8周龄Balb/c小鼠骨髓单核细胞,脂多糖及干扰素γ诱导为M1巨噬细胞,分M1(M1)、MT(M1+rhTSG-6)、MM(M1+MSCs)、MMsi(M1+MSCs+TSG-6siRNA)4组共培养3 d,收集巨噬细胞和上清液,流式检测巨噬细胞表型比例;ELISA检测炎症因子白细胞介素1β、白细胞介素12、肿瘤坏死因子α、转化生长因子β1、白细胞介素10、TSG-6水平;Western blot检测巨噬细胞极化相关p/t-STAT1/3/6及TSG-6水平;RT-PCR检测精氨酸酶1、白细胞介素10、诱导型一氧化氮合酶、肿瘤坏死因子α的mRNA表达。②阻断M1表面CD44:分M1(M1)、MT(M1+rhTSG-6)、bMT(M1+TSG-6+blkCD44组)、MM(M1+MSCs)、bMM(M1+MSC+blkCD44组)5组共培养3 d,Western blot检测各组细胞CD44、p/t-STAT1/3/6水平;RT-PCR检测CD44、STAT1/3/6 mRNA和目的基因表达水平;流式分析阻断CD44后TSG-6黏附阳性M1巨噬细胞。结果与结论:小鼠M1巨噬细胞加入rhTSG-6或与人脐血间充质干细胞共培养后观察到:①M1细胞减少、M2细胞增加、M1/M2下降(P<0.05);②促炎症因子肿瘤坏死因子α、白细胞介素1β和白细胞介素12水平降低(P<0.05),抗炎症因子白细胞介素10、转化生长因子β1和TSG-6水平升高(P<0.0BACKGROUND:One of the main mechanisms for stem cell transplantation to treat diseases is regulating inflammatory response in a manner of paracrine or distant secretion.Transplanted stem cells secrete a large number of biologically molecules,such as transforming growth factor beta,prostaglandin E2,interleukin 10,and tumor necrosis factorαstimulating gene-6 protein(TSG-6),which affect the conversion of monocyte/macrophages from the proinflammatory M1 phenotype to the anti-inflammatory M2 phenotype,and coordinating the balance of pro-inflammatory and anti-inflammatory factors.The mechanism by which stem cell secreting factors promote the conversion of monocyte/macrophages M1/M2 subtypes is not clear.OBJECTIVE:To investigate the effects of biologically active molecule secreted by human umbilical cord blood mesenchymal stem cells TSG-6 on the conversions of phenotype and inflammatory factors of mice bone marrow-derived macrophages,and research into the mechanism of TSG-6 protein-mediated M2 polarization in macrophages.METHODS:(1)Bone marrow derived monocytes aseptically extracted from 6-8-week-old Balb/c mice were differentiated into M1 macrophages with lipopolysaccharide and interferon-γ.M1 macrophages were divided into four groups:M1(M1),MT(M1+rhTSG-6),MM(M1+MSCs),and MMsi(M1+MSCs+TSG-6 siRNA).After co-culture for 3 days,cells and supernatant fluid were collected.The macrophages M1/M2 ratio were measured by flow cytometry.The concentrations of inflammatory factors interleukin 1β,interleukin 12,tumor necrosis factorα,transforming growth factorβ1,interleukin 10,and TSG-6 were measured by ELISA.Inside the macrophage,p/t-STAT1/3/6 and TSG-6 levels were measured by western blot assay.mRNA expression levels of arginase 1,interleukin 10,inducible nitric oxide synthase,and tumor necrosis factor alpha were measured by RT-PCR.(2)After blocking CD44,M1 macrophages were divided into five groups:M1(M1),MT(M1+rhTSG-6),bMT(M1+TSG-6+blkCD44),MM(M1+MSCs),and bMM(M1+MSC+blkCD44).After co-culture for 3 days,cells were collected t

关 键 词：炎症 人脐血间充质干细胞 单核/巨噬细胞 M1/M2 TSG-6 CD44 STAT 

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