中国组织工程研究

中国组织工程研究 ›› 2022, Vol. 26 ›› Issue (13): 2012-2019.doi: 10.12307/2022.326

• 脐带脐血干细胞 umbilical cord blood stem cells • 上一篇    下一篇

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

赵吉玲，彭  漪，彭智勇，余国龙   

  1. 中南大学湘雅医院心内科，湖南省长沙市   410008
  • 收稿日期:2020-03-06 修回日期:2020-03-12 接受日期:2020-12-14 出版日期:2022-05-08 发布日期:2021-12-20
  • 通讯作者: 余国龙，博士，教授，中南大学湘雅医院心内科，湖南省长沙市 410008
  • 作者简介:赵吉玲，女，1992年生，湖北省恩施市人，土家族，2019年中南大学湘雅医学院毕业，硕士，医师，主要从事干细胞治疗心肌梗死的基础医学研究。
  • 基金资助:
    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

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

Zhao Jiling, Peng Yi, Peng Zhiyong, Yu Guolong   

  1. Department of Cardiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
  • Received:2020-03-06 Revised:2020-03-12 Accepted:2020-12-14 Online:2022-05-08 Published:2021-12-20
  • Contact: Yu Guolong, MD, Professor, Department of Cardiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
  • About author:Zhao Jiling, Master, Physician, Department of Cardiology, Xiangya Hospital, Central South University, Changsha 410008, Hunan Province, China
  • Supported by:
    National Natural Science Foundation of China, No. 81570266 (to YGL); Hunan Provincial Natural Science Foundation Key Project, No. 2015SK2025 (to YGL)

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摘要：

文题释义：
肿瘤坏死因子α-刺激基因-6蛋白(TSG-6)：是一种分子质量为35-38 kD的糖蛋白，可由活化的间充质干细胞分泌，有抗炎蛋白之称，在心肌梗死、脑梗死及动脉粥样硬化等动物模型中被证明有抗炎及组织保护作用。由促炎性细胞因子如白细胞介素1β、干扰素γ、肿瘤坏死因子α、脂多糖等诱导TSG-6表达，但是抑炎性的细胞因子如白细胞介素4或白细胞介素10则抑制TSG-6表达。在观测一些疾病模型中发现间充质干细胞以TSG-6依赖性方式调节巨噬细胞表型向M2型转化、调控炎症反应、增强组织修复和防止过度组织纤维化，这可能是干细胞促进单核巨噬细胞亚型转化的机制之一。
巨噬细胞亚型：巨噬细胞M1和M2特征之间的转换被称为“极化”。巨噬细胞极化是一种过程，在特定的组织和特定的微环境中，巨噬细胞表现出特定的表型和对微环境刺激的功能反应，目前已经认识到2个主要巨噬细胞亚群包括经典活化的炎性(M1)和可活化的抗炎(M2)巨噬细胞，M2巨噬细胞可以进一步分为M2a，M2b，M2c和M2d亚型，最近也有将M1向M2转化的中间状态细胞称为M3的说法，其性质有待进一步探究。


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 factor α stimulating 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 < 0.05)；③p/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mRNA表达降低，白细胞介素10 mRNA和精氨酸酶1 mRNA表达升高(P < 0.05)。阻断M1表面CD44后观察到：①TSG-6黏附阳性细胞比例降低(P < 0.05)；②CD44蛋白及mRNA表达下降(P < 0.05)；③p/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 mRNA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。
缩略语：人脐血间充质干细胞：human umbilical cord blood-mesenchymal stem cells，hUCB-MSCs；肿瘤坏死因子α-刺激基因-6蛋白：tumor necrosis factor α stimulating gene-6 protein，TSG-6

https://orcid.org/0000-0002-1130-6203 (赵吉玲) 

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

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

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 pro-inflammatory 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 1beta 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.

Key words: inflammation, human umbilical cord blood mesenchymal stem cells, mononcyte/macrophages, M1/M2, TSG-6, CD44, STAT

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