【摘要】：本文基于對(duì)人外周血單核細(xì)胞來(lái)源的耐受型樹(shù)突狀細(xì)胞(tolerogenic dendritic cells, tDC)的免疫耐受功能和1-磷酸鞘氨醇(Sphingosine-1-phosphate,S1P)的生理作用進(jìn)行研究,首次闡述了S1P在體外對(duì)人tDC免疫學(xué)功能的影響。 未成熟樹(shù)突狀細(xì)胞(immature dendritic cells, iDC)可作為一種發(fā)揮免疫耐受功能的DC在移植物抗宿主病中發(fā)揮重要作用。但是越來(lái)越多的研究者發(fā)現(xiàn),tDC在移植物排斥和多種自身免疫性疾病中能更好地發(fā)揮耐受功能。本實(shí)驗(yàn)用流式細(xì)胞術(shù)和RT-PCR的方法分別檢測(cè)tDC CD209、HLA-DR、CD80、CD83、CD86幾種表型分子的表達(dá)以及細(xì)胞內(nèi)IL-10、TGF-β、Fas-L、IDO、IL-12p40以及TNF-a mRNA的表達(dá)。結(jié)果顯示該tDC高表達(dá)HLA-DR、DC標(biāo)志CD209、以及發(fā)揮免疫抑制功能的IL-10、TGF-β、Fas-L、IDO的mRNA,低表達(dá)成熟標(biāo)志CD83和共刺激分子CD80、CD86以及兩種炎性細(xì)胞因子IL-12p40和TNF-a mRNA。在表型上屬于一種未成熟DC。 目前鑒于對(duì)S1P影響免疫系統(tǒng)功能研究的逐步深入,S1P對(duì)免疫系統(tǒng)中重要的細(xì)胞亞群tDC的影響引起了我們的興趣。本實(shí)驗(yàn)用RT-PCR檢測(cè)的方法證明tDC表達(dá)S1P受體1,2,5,不表達(dá)受體3和4且S1P受體1和受體2 mRNA的表達(dá)均明顯高于S1P受體5。流式方法檢測(cè)出10-5M的S1P會(huì)引起tDC細(xì)胞內(nèi)明顯的Ca2+流的增加。Ca2+的變化提示我們S1P確實(shí)能與tDC表面的受體結(jié)合導(dǎo)致tDC的活化。S1P作用于tDC后,其表型和炎性細(xì)胞因子IL-12p40和TNF-a mRNA的表達(dá)無(wú)明顯變化,仍處于未成熟狀態(tài),卻上調(diào)了發(fā)揮免疫抑制功能的IL-10、TGF-β和Fas-L mRNA的表達(dá)。因此進(jìn)一步用Western Blot方法證明了S1P會(huì)導(dǎo)致細(xì)胞內(nèi)信號(hào)蛋白ERK磷酸化水平發(fā)生變化：S1P在一定濃度范圍內(nèi)增強(qiáng)ERK磷酸化水平,隨濃度升高ERK磷酸化水平有減弱的趨勢(shì)。CBA方法檢測(cè)出當(dāng)S1P和LPS共同作用于tDC時(shí),S1P會(huì)濃度依賴(lài)性地抑制由LPS引起的tDC分泌IL-10的能力。本研究針對(duì)S1P對(duì)tDC功能影響的結(jié)果可為今后tDC在移植物排斥和自身免疫性疾病中的細(xì)胞治療和S1P類(lèi)似物免疫抑制劑的化學(xué)治療提供實(shí)驗(yàn)依據(jù)和新的治療方向。
[Abstract]:The effects of S1P on the immunological function of human tDC in vitro were studied based on the immune tolerance function of human peripheral blood monocyte derived tolerant dendritic cells (tolerogenic dendritic cells, tDC) and the physiological function of sphingosine 1-phosphate (Sphingosine-1-phosphate,S1P). Immature dendritic cells (immature dendritic cells, iDC) may play an important role in graft-versus-host disease (GVHD) as an immune tolerant DC. However, more and more researchers have found that tDC plays a better role in tolerance in graft rejection and many autoimmune diseases. The expression of several phenotypic molecules of tDC CD209,HLA-DR,CD80,CD83,CD86 and the expression of IL-10,TGF- 尾, Fas-L,IDO,IL-12p40 and TNF-a mRNA in cells were detected by flow cytometry and RT-PCR, respectively. The results showed that the high expression of HLA-DR,DC marker CD209, and the immunosuppressive IL-10,TGF- 尾, the low expression of mRNA, mature marker CD83 and costimulatory molecule CD80,CD86, and the two inflammatory cytokines IL-12p40 and TNF-a mRNA. were found in this tDC. Phenotypic belonging to an immature DC. At present, the influence of S1P on the function of immune system has attracted our interest, and the influence of S1P on the important cell subsets of the immune system (tDC) has aroused our interest. The expression of S1P receptor 1 and S1P receptor 2 mRNA in tDC was significantly higher than that in S1P receptor 5, and the expression of S1P receptor 1 and S1P receptor 2 mRNA was significantly higher than that of S1P receptor 5. Flow cytometry showed that 10-5M S1P could increase the Ca2 flow in tDC cells. The changes of Ca2 suggest that S1P can bind to the receptor on tDC surface and lead to the activation of tDC. S1P acts on tDC. Its phenotype and the expression of inflammatory cytokines, IL-12p40 and TNF-a mRNA, remained immature, but up-regulated the expression of IL-10,TGF- 尾 and Fas-L mRNA, which played an immunosuppressive role. It was further proved by Western Blot method that S1P could cause changes in intracellular signal protein ERK phosphorylation level: S1P enhanced ERK phosphorylation level in a certain concentration range. CBA method showed that when S1P and LPS acted on tDC, S1P inhibited the IL-10 secretion of tDC induced by LPS in a concentration-dependent manner. The results of this study on the effect of S1P on tDC function may provide experimental basis and new therapeutic direction for cell therapy of tDC in graft rejection and autoimmune diseases and chemotherapy of S1P analogues immunosuppressant.
【學(xué)位授予單位】：華東師范大學(xué)
【學(xué)位級(jí)別】：碩士
【學(xué)位授予年份】：2011
【分類(lèi)號(hào)】：R392

【共引文獻(xiàn)】

相關(guān)期刊論文 前10條

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本文編號(hào)：2283708