间充质干细胞调控选择性活化树突状细胞发育

发布时间：2018-11-17 16:09

【摘要】： 微环境与免疫细胞的发育分化和功能密切相关,选择性活化树突状细胞(AADC)作为一新型具有独特免疫耐受作用的DC细胞亚群,随着移植医学的不断发展,正日益成为研究的热点。DC的发育受控于造血微环境,间充质干细胞(MSC)作为造血微环境的原始细胞,其调控AADC的发育、功能与作用机制均未见报道。本研究就此展开深入探讨。 首先,利用MSC与CD34+细胞共培养诱导体系,获得DC(即MSC-DC)。从形态、表型和功能三方面与常规诱导获得的未成熟DC、成熟DC和IL-10诱导获得的AADC进行比较鉴定。结果表明,在MSC与CD34+细胞共培养诱导体系中获得的MSC-DC具有细小的伸出突起,细胞低表达CD80、CD86、HLA-DR等共刺激分子;刺激异体T细胞增殖功能明显下降;且细胞在Matrigel上可形成血管样结构,呈网络状生长;呈现典型的AADC特征。细胞不能吞噬FITC-Dextran,表达趋化因子受体CCR7和CXCR4,具有明显的成熟DC特性。 其次,就MSC对AADC发育的作用机制进行研究。选择与DC发育密切相关的Notch信号通路作为切入点,利用RNA干扰技术,选择性敲除MSC上Notch配体Jagged1,探讨MSC调控AADC生成的作用机制。结果表明,Real-time PCR检测MSC与CD34+细胞共培养前后Notch受体与配体均发生显著改变。进一步,利用GFP慢病毒转染体系获得选择性敲除Jagged1的MSC(即Jag1-MSC)。与Jag1-MSC共培养获得的DC(即Jag1-MSC-DC),Notch信号通路活化标志转录因子Hes1表达降低,共刺激分子CD86、CD83、HLA-DR表达上升,Th1类细胞因子IL-12表达升高,而Th2类细胞因子IL-10降低,同时刺激T细胞增殖能力有所增加,说明Notch通路在AADC的诱导分化中发挥重要作用。另外, Notch通路与对干细胞的增殖分化起重要作用的MAPK通路是否存在交叉进行了初步研究。Western结果显示,MAPK中的p-ERK蛋白对应的表达增加,提示MAPK通路在Notch通路阻断后代偿性表达升高。 最后,探讨AADC发育对MSC生物学特性的影响。结果显示, MSC形态未发生明显变化,细胞表达CD29、CD73、HLA-ABC及CD86有所降低,细胞失去成脂肪能力,而TGF-β、G-CSF、M-CSF、GM-CSF等细胞因子表达升高,提示在DC刺激下,MSC进一步加强其免疫调节特性。 以上结果首次证明MSC通过Notch信号通路调控CD34+细胞发育分化为AADC,为进一步了解DC的发育分化及解决临床移植排斥等问题提供有效的新思路和理论实验基础。
[Abstract]:Microenvironment is closely related to the development, differentiation and function of immune cells. Selective activation of dendritic cells (AADC), as a new type of DC cell subsets with unique immune tolerance, develops with the development of transplantation medicine. The development of DC is controlled by hematopoietic microenvironment. As a primitive cell of hematopoietic microenvironment, the development, function and mechanism of mesenchymal stem cell (MSC) have not been reported. This study is discussed in depth. Firstly, DC (MSC-DC) was obtained by co-culture of MSC and CD34 cells. Morphological, phenotypic and functional aspects were compared with immature DC, DC and AADC induced by IL-10. The results showed that the MSC-DC obtained in the co-culture system of MSC and CD34 cells had small protrusions, low expression of CD80,CD86,HLA-DR and other costimulatory molecules, and significantly decreased the proliferation of allogeneic T cells. The cells formed vascular-like structure on Matrigel, and showed typical characteristics of AADC. The expression of chemokine receptors CCR7 and CXCR4, by FITC-Dextran, could not be phagocytized by cells. Secondly, the mechanism of MSC on AADC development was studied. The Notch signaling pathway closely related to the development of DC was selected as the starting point. The mechanism of MSC regulating AADC production was investigated by using RNA interference technique and selectively knockout the Notch ligand Jagged1, on MSC. The results showed that the Notch receptor and ligand of MSC and CD34 cells were significantly changed before and after co-culture by Real-time PCR. Furthermore, the selective knockout MSC (Jag1-MSC) of Jagged1 was obtained by using GFP lentivirus transfection system. DC co-cultured with Jag1-MSC (that is, Jag1-MSC-DC), Notch signal pathway activation marker transcription factor Hes1 expression decreased, costimulatory molecule CD86,CD83,HLA-DR expression increased, Th1 cytokine IL-12 expression increased. The decrease of Th2 cytokine IL-10 and the increase of T cell proliferation suggest that the Notch pathway plays an important role in the induction and differentiation of AADC. In addition, whether there is a cross between the Notch pathway and the MAPK pathway, which plays an important role in the proliferation and differentiation of stem cells, was preliminarily studied. The Western results showed that the corresponding expression of p-ERK protein in MAPK increased. The results suggest that the compensatory expression of MAPK pathway is increased after the Notch pathway is blocked. Finally, the effects of AADC development on the biological characteristics of MSC were discussed. The results showed that the morphology of MSC did not change significantly, the expression of CD29,CD73,HLA-ABC and CD86 decreased, and the cells lost the ability of adipogenesis, while the expression of TGF- 尾, G-CSFM-GM-CSF and other cytokines increased. The results suggest that MSC can further enhance its immunomodulatory properties under DC stimulation. The above results demonstrate for the first time that MSC regulates the development and differentiation of CD34 cells into AADC, through Notch signaling pathway, which provides an effective theoretical and experimental basis for further understanding the development and differentiation of DC and solving the problems of clinical transplant rejection.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R392

【参考文献】

中国期刊全文数据库 前1条

1 江小霞;苏永锋;李秀森;张毅;吴英;毛宁;;胎儿心脏黏附细胞具有类似间充质祖细胞特征(英文)[J];中国实验血液学杂志;2006年06期

，

本文编号：2338391