1-磷酸鞘氨醇对人耐受型树突状细胞功能影响的初步研究
发布时间：2018-10-20 16:25
【摘要】：本文基于对人外周血单核细胞来源的耐受型树突状细胞(tolerogenic dendritic cells, tDC)的免疫耐受功能和1-磷酸鞘氨醇(Sphingosine-1-phosphate,S1P)的生理作用进行研究,首次阐述了S1P在体外对人tDC免疫学功能的影响。 未成熟树突状细胞(immature dendritic cells, iDC)可作为一种发挥免疫耐受功能的DC在移植物抗宿主病中发挥重要作用。但是越来越多的研究者发现,tDC在移植物排斥和多种自身免疫性疾病中能更好地发挥耐受功能。本实验用流式细胞术和RT-PCR的方法分别检测tDC CD209、HLA-DR、CD80、CD83、CD86几种表型分子的表达以及细胞内IL-10、TGF-β、Fas-L、IDO、IL-12p40以及TNF-a mRNA的表达。结果显示该tDC高表达HLA-DR、DC标志CD209、以及发挥免疫抑制功能的IL-10、TGF-β、Fas-L、IDO的mRNA,低表达成熟标志CD83和共刺激分子CD80、CD86以及两种炎性细胞因子IL-12p40和TNF-a mRNA。在表型上属于一种未成熟DC。 目前鉴于对S1P影响免疫系统功能研究的逐步深入,S1P对免疫系统中重要的细胞亚群tDC的影响引起了我们的兴趣。本实验用RT-PCR检测的方法证明tDC表达S1P受体1,2,5,不表达受体3和4且S1P受体1和受体2 mRNA的表达均明显高于S1P受体5。流式方法检测出10-5M的S1P会引起tDC细胞内明显的Ca2+流的增加。Ca2+的变化提示我们S1P确实能与tDC表面的受体结合导致tDC的活化。S1P作用于tDC后,其表型和炎性细胞因子IL-12p40和TNF-a mRNA的表达无明显变化,仍处于未成熟状态,却上调了发挥免疫抑制功能的IL-10、TGF-β和Fas-L mRNA的表达。因此进一步用Western Blot方法证明了S1P会导致细胞内信号蛋白ERK磷酸化水平发生变化：S1P在一定浓度范围内增强ERK磷酸化水平,随浓度升高ERK磷酸化水平有减弱的趋势。CBA方法检测出当S1P和LPS共同作用于tDC时,S1P会浓度依赖性地抑制由LPS引起的tDC分泌IL-10的能力。本研究针对S1P对tDC功能影响的结果可为今后tDC在移植物排斥和自身免疫性疾病中的细胞治疗和S1P类似物免疫抑制剂的化学治疗提供实验依据和新的治疗方向。
[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.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
[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.
【学位授予单位】：华东师范大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R392
【共引文献】
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