【摘要】： 微環(huán)境與免疫細(xì)胞的發(fā)育分化和功能密切相關(guān),選擇性活化樹突狀細(xì)胞(AADC)作為一新型具有獨(dú)特免疫耐受作用的DC細(xì)胞亞群,隨著移植醫(yī)學(xué)的不斷發(fā)展,正日益成為研究的熱點(diǎn)。DC的發(fā)育受控于造血微環(huán)境,間充質(zhì)干細(xì)胞(MSC)作為造血微環(huán)境的原始細(xì)胞,其調(diào)控AADC的發(fā)育、功能與作用機(jī)制均未見報(bào)道。本研究就此展開深入探討。 首先,利用MSC與CD34+細(xì)胞共培養(yǎng)誘導(dǎo)體系,獲得DC(即MSC-DC)。從形態(tài)、表型和功能三方面與常規(guī)誘導(dǎo)獲得的未成熟DC、成熟DC和IL-10誘導(dǎo)獲得的AADC進(jìn)行比較鑒定。結(jié)果表明,在MSC與CD34+細(xì)胞共培養(yǎng)誘導(dǎo)體系中獲得的MSC-DC具有細(xì)小的伸出突起,細(xì)胞低表達(dá)CD80、CD86、HLA-DR等共刺激分子;刺激異體T細(xì)胞增殖功能明顯下降;且細(xì)胞在Matrigel上可形成血管樣結(jié)構(gòu),呈網(wǎng)絡(luò)狀生長;呈現(xiàn)典型的AADC特征。細(xì)胞不能吞噬FITC-Dextran,表達(dá)趨化因子受體CCR7和CXCR4,具有明顯的成熟DC特性。 其次,就MSC對(duì)AADC發(fā)育的作用機(jī)制進(jìn)行研究。選擇與DC發(fā)育密切相關(guān)的Notch信號(hào)通路作為切入點(diǎn),利用RNA干擾技術(shù),選擇性敲除MSC上Notch配體Jagged1,探討MSC調(diào)控AADC生成的作用機(jī)制。結(jié)果表明,Real-time PCR檢測(cè)MSC與CD34+細(xì)胞共培養(yǎng)前后Notch受體與配體均發(fā)生顯著改變。進(jìn)一步,利用GFP慢病毒轉(zhuǎn)染體系獲得選擇性敲除Jagged1的MSC(即Jag1-MSC)。與Jag1-MSC共培養(yǎng)獲得的DC(即Jag1-MSC-DC),Notch信號(hào)通路活化標(biāo)志轉(zhuǎn)錄因子Hes1表達(dá)降低,共刺激分子CD86、CD83、HLA-DR表達(dá)上升,Th1類細(xì)胞因子IL-12表達(dá)升高,而Th2類細(xì)胞因子IL-10降低,同時(shí)刺激T細(xì)胞增殖能力有所增加,說明Notch通路在AADC的誘導(dǎo)分化中發(fā)揮重要作用。另外, Notch通路與對(duì)干細(xì)胞的增殖分化起重要作用的MAPK通路是否存在交叉進(jìn)行了初步研究。Western結(jié)果顯示,MAPK中的p-ERK蛋白對(duì)應(yīng)的表達(dá)增加,提示MAPK通路在Notch通路阻斷后代償性表達(dá)升高。 最后,探討AADC發(fā)育對(duì)MSC生物學(xué)特性的影響。結(jié)果顯示, MSC形態(tài)未發(fā)生明顯變化,細(xì)胞表達(dá)CD29、CD73、HLA-ABC及CD86有所降低,細(xì)胞失去成脂肪能力,而TGF-β、G-CSF、M-CSF、GM-CSF等細(xì)胞因子表達(dá)升高,提示在DC刺激下,MSC進(jìn)一步加強(qiáng)其免疫調(diào)節(jié)特性。 以上結(jié)果首次證明MSC通過Notch信號(hào)通路調(diào)控CD34+細(xì)胞發(fā)育分化為AADC,為進(jìn)一步了解DC的發(fā)育分化及解決臨床移植排斥等問題提供有效的新思路和理論實(shí)驗(yàn)基礎(chǔ)。
[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.
【學(xué)位授予單位】：中國人民解放軍軍事醫(yī)學(xué)科學(xué)院
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2009
【分類號(hào)】：R392

【參考文獻(xiàn)】

中國期刊全文數(shù)據(jù)庫 前1條

1 江小霞;蘇永鋒;李秀森;張毅;吳英;毛寧;;胎兒心臟黏附細(xì)胞具有類似間充質(zhì)祖細(xì)胞特征(英文)[J];中國實(shí)驗(yàn)血液學(xué)雜志;2006年06期

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本文編號(hào)：2338391