免疫抑制剂他克莫司诱导的耐受性树突状细胞生物学特性及功能的初步研究

发布时间：2018-04-11 14:51

本文选题：耐受性树突状细胞 + 他克莫司　；参考：《复旦大学》2014年硕士论文

【摘要】：本文基于对人外周血单核细胞来源的耐受性树突状细胞(tolerogenic dendritic cells, tDC)的生物学特性和免疫学功能,建立免疫抑制剂他克莫司诱导人来源的耐受性树突状细胞的方法,并分析其生物学特性及免疫抑制功能。树突状细胞(dendritic cell, DC)是体内最重要的抗原提呈细胞,也是唯一可以诱导幼稚T淋巴细胞(naive T cells)活化的专职抗原递呈细胞,在免疫调节中有着免疫应答和免疫耐受的双重作用。近年来,随着对DC异质性的深入研究,DC在激活免疫应答的同时也可负向调节免疫应答强度,诱导免疫耐受这一特性越来越引起人们重视。耐受性树突状细胞具有负向免疫调节功能,在体内外诱导T细胞的低反应性,并诱导调节性T细胞(regulatory T cells, Treg)的产生,参与免疫耐受的形成,从而能够治疗多种疾病如器官移植、自身免疫性疾病等应用于临床。但体内DC的含量极微,其数量不及外周血白细胞数量的1%,不能满足临床试验需求,如何在体外诱生DC并维持其处于负向调节免疫反应的功能状态成为其应用于临床的关键。有文献报道：药物如免疫抑制剂(地塞米松、环孢素A、雷帕霉素、脱氧精胍菌素和麦考酚吗乙酯等)、阿司匹林、维生素D等以及细胞因子如IL-10、TGF-p等均可刺激tDC产生。本研究建立了免疫抑制剂他克莫司诱导人来源的耐受性树突状细胞的方法,并且分析和证实了其生物学特性及免疫抑制功能。本实验从人外周血分离单个核细胞(PBMC), PBMC在cGMP级培养液CellGro DC中添加GM-CSF和IL-4诱导培养树突状细胞(DC),培养过程中添加免疫抑制药他克莫司获得tDC。然后采用流式细胞术检测他克莫司诱导的tDCCD209、HLA-DR、CD80、CD83、CD86几种表型分子的表达及其细胞活性。结果显示,他克莫司诱导的tDC具有典型的耐受性细胞表型：高表达DC标志CD209,低表达共刺激分子CD80、CD86、CD83以及HLA-DR,他克莫司不影响单核细胞向DC的分化。同时,本实验还采用Annexin V/PI法检测了他克莫司诱导的tDC的活性,结果显示,该法诱导的tDC具有较高的活性。在对他克莫司诱导的tDC的免疫学功能的研究中,通过CFSE增殖实验检测其调节异源CD4+效应T细胞的作用,同时比较了他克莫司诱导的tDC与Il-10/TGF-β、地塞米松、环孢素A诱导的tDC的耐受功能。功能分析显示,与未加药的相比,药物地塞米松、环孢霉素A或他克莫司刺激的tDC具有对异源细胞的低反应性,不能诱导异源CD4+T细胞增殖。而他克莫司诱导的tDC更有效地抑制成熟DC引起的混合淋巴细胞反应。本实验也验证了此方法诱导培养的tDC冻存复苏后仍具有表型和功能上的稳定性。虽然有研究报道显示用地塞米松、IL-10、TGF-β等能诱导产生tDC,但对诱导产生的tDC调节T细胞的能力很少有研究,本研究建立了他克莫司诱导tDC的培养方法,同时研究了用此法诱导的tDC的免疫学功能,及冻存复苏后的tDC的生物学特性及功能,证实免疫抑制药物他克莫司诱导培养的tDC具有耐受功能,为其在自身免疫性疾病、器官移植耐受等方面的治疗提供了科学的实验室依据,具有重要的现实临床意义。
[Abstract]:This paper on human peripheral blood monocyte derived tolerogenic dendritic cells (tolerogenic dendritic cells, based on tDC) biological characteristics and immune function, establishing method of tacrolimus induced human derived tolerogenic dendritic cells, and analyze the characteristics of biology and immune suppression function. Dendritic cells (dendritic cell, DC) are the most potent antigen-presenting cells, and only can induce naive T lymphocytes (naive T cells) activation of antigen presenting cells, has a dual role of immune response and immune tolerance in immune regulation. In recent years, with in-depth study of the heterogeneity of DC, DC in the activation of immune response at the same time also can negatively regulate the intensity of immune response, immune tolerance induced by the characteristic of more and more attention. Tolerogenic dendritic cells had the ability of negative immune modulation in vivo. Low response induced by T cells, and induce regulatory T cells (regulatory T cells, Treg) which involved in the formation of immune tolerance, which can treat various diseases such as organ transplantation, autoimmune disease in clinical application. But the low content of DC in vivo, the number is less than the number of peripheral white blood cell 1%, can not meet the needs of clinical trials, how to maintain and the negative functions of state regulation of the immune response becomes the key to clinical application of in vitro induced DC. It is reported that drugs such as immunosuppressive drugs (dexamethasone, cyclosporin A, rapamycin, deoxyspergualin and mycophenolate mofetil ethyl), aspirin, vitamin D and other cytokines such as IL-10, TGF-p can stimulate the production of tDC. The method was established in this study of tacrolimus induced human derived tolerogenic dendritic cells, and analyzed and confirmed The biological characteristics and its immunosuppressive function. Mononuclear cells were isolated from human peripheral blood in this experiment (PBMC), PBMC medium CellGro DC add GM-CSF and IL-4 induced by dendritic cells cultured in cGMP (DC), in the training process to add immunosuppressive drug tDC. and tacrolimus induced by tacrolimus was detected by flow cytometry tDCCD209, HLA-DR, CD80, CD83, CD86 expression of several phenotypic molecules and cell activity. The results showed that tacrolimus induced tDC cell tolerance phenotype is typical: high expression of DC markers of CD209, low expression of costimulatory molecules CD80, CD86, CD83 and HLA-DR, tacrolimus does not affect mononuclear cells to the differentiation of DC. At the same time, this experiment also used Annexin V/PI method to detect tacrolimus induced tDC activity. The results show that this method has higher activity induced by tDC. In immunology of tacrolimus induced tDC The study, the regulation of heterologous CD4+ effector T cell function was detected by CFSE proliferation assay, and compares tDC and Il-10/TGF- beta, tacrolimus induced by dexamethasone and cyclosporin A induced tDC tolerance function. Functional analysis shows that, compared with no drug drug dexamethasone and cyclosporin or tacrolimus A stimulating hormone the tDC has a low responsiveness to heterologous cells, proliferation of heterologous cells induced by CD4+T. The mixed lymphocyte reaction and tacrolimus induced tDC effectively inhibit maturation induced by DC. This experiment also verified this method of induction of tDC after cryopreservation has stability of phenotype and function. Although there are reports on display dexamethasone, IL-10, TGF- P can induce tDC, but the ability to induce tDC regulatory T cells, there is little research, based on the cultivation of tacrolimus induced by tDC Method, immunological function was also studied by this method induced tDC, and cryopreserved tDC biological characteristics and function, confirmed the immunosuppressive drugs tacrolimus induced by tDC with tolerance for its function in autoimmune diseases, provide experimental basis for the scientific treatment of organ transplantation tolerance and other aspects, has the reality an important clinical significance.

【学位授予单位】：复旦大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R96

【共引文献】