【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-6protein,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+rh TSG-6)、MM(M1+MSCs)、MMsi(M1+MSCs+TSG-6si RNA)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m RNA表达。(2)阻断M1表面CD44:分M1(M1)、MT(M1+rh TSG-6)、b MT(M1+TSG-6+blk CD44组)、MM(M1+MSCs)、b MM(M1+MSC+blk CD44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 m RNA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rh TSG-6或与人脐血间充质干细胞共培养后观察到:(1)M1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 <0.05);(3)p/t-STAT1水平降低、p/t-STAT3/6水平升高(P <0.05);(4)肿瘤坏死因子α和诱导型一氧化氮合酶m RNA表达降低,白细胞介素10 m RNA和精氨酸酶1 m RNA表达升高(P <0.05)。阻断M1表面CD44后观察到:(1)TSG-6黏附阳性细胞比例降低(P <0.05);(2)CD44蛋白及mRNA表达下降(P <0.05);(3)p/t-STAT1、肿瘤坏死因子α及诱导型一氧化氮合酶mRNA表达升高(P <0.05),p/t-STAT3/6 mRNA、白细胞介素10及精氨酸酶1 m RNA表达降低(P <0.01)。因此,该研究阐述了人脐血间充质干细胞分泌的TSG-6以CD44依赖的方式影响STAT1/3/6信号通路介导巨噬细胞M1向M2亚型转化及炎症调节。更多还原

【Abstract】 BACKGROUND: 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 factor α stimulating 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 factor α, transforming growth factor β1, 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 to perform western blot assay for the concentrations of CD44 and p/t-STAT1/3/6. RT-PCR was conducted for the expression of CD44 and STAT1/3/6 mRNA and target genes. TSG-6 adhered M1 macrophages were measured by flow cytometry.RESULTS AND CONCLUSION: Mouse M1 macrophages co-cultured with rhTSG-6 or human cord blood mesenchymal stem cells showed:(1) M1 cells decreased,M2 cells increased, and the ratio of M1/M2 decreased(P < 0.05).(2) The pro-inflammatory factors tumor necrosis factor alpha, interleukin 1 beta and interleukin 12 decreased(P < 0.05), and the levels of anti-inflammatory factors interleukin 10, transforming growth factor beta1 and TSG-6 increased(P < 0.05).(3) p/t-STAT1 levels decreased and p/t-STAT3/6 level increased(P < 0.05).(4) Tumor necrosis factor alpha and inducible nitric oxide synthase mRNA expression decreased, and interleukin 10 mRNA and arginase 1 mRNA expression increased(P < 0.05). After blocking CD44:(1) TSG-6 adhered macrophages decreased(P < 0.05).(2) CD44 protein and mRNA expression decreased(P < 0.05).(3) The mRNA expression of p/t-STAT1, tumor necrosis factor alpha, and inducible nitric oxide synthase significantly increased(P < 0.05), and the mRNA expression of p/t-STAT3/6, interleukin 10, and arginase 1(P < 0.01). Therefore, human umbilical cord blood mesenchymal stem cells secreting TSG-6 mediates M1 to M2 conversion and inflammatory regulation in macrophages by affecting STAT 1/3/6 signaling pathway in a CD44-dependent manner.更多还原