本文選題：耐受性樹突狀細胞 + 他克莫司　； 參考：《復(fù)旦大學(xué)》2014年碩士論文

【摘要】：本文基于對人外周血單核細胞來源的耐受性樹突狀細胞(tolerogenic dendritic cells, tDC)的生物學(xué)特性和免疫學(xué)功能,建立免疫抑制劑他克莫司誘導(dǎo)人來源的耐受性樹突狀細胞的方法,并分析其生物學(xué)特性及免疫抑制功能。樹突狀細胞(dendritic cell, DC)是體內(nèi)最重要的抗原提呈細胞,也是唯一可以誘導(dǎo)幼稚T淋巴細胞(naive T cells)活化的專職抗原遞呈細胞,在免疫調(diào)節(jié)中有著免疫應(yīng)答和免疫耐受的雙重作用。近年來,隨著對DC異質(zhì)性的深入研究,DC在激活免疫應(yīng)答的同時也可負向調(diào)節(jié)免疫應(yīng)答強度,誘導(dǎo)免疫耐受這一特性越來越引起人們重視。耐受性樹突狀細胞具有負向免疫調(diào)節(jié)功能,在體內(nèi)外誘導(dǎo)T細胞的低反應(yīng)性,并誘導(dǎo)調(diào)節(jié)性T細胞(regulatory T cells, Treg)的產(chǎn)生,參與免疫耐受的形成,從而能夠治療多種疾病如器官移植、自身免疫性疾病等應(yīng)用于臨床。但體內(nèi)DC的含量極微,其數(shù)量不及外周血白細胞數(shù)量的1%,不能滿足臨床試驗需求,如何在體外誘生DC并維持其處于負向調(diào)節(jié)免疫反應(yīng)的功能狀態(tài)成為其應(yīng)用于臨床的關(guān)鍵。有文獻報道：藥物如免疫抑制劑(地塞米松、環(huán)孢素A、雷帕霉素、脫氧精胍菌素和麥考酚嗎乙酯等)、阿司匹林、維生素D等以及細胞因子如IL-10、TGF-p等均可刺激tDC產(chǎn)生。本研究建立了免疫抑制劑他克莫司誘導(dǎo)人來源的耐受性樹突狀細胞的方法,并且分析和證實了其生物學(xué)特性及免疫抑制功能。本實驗從人外周血分離單個核細胞(PBMC), PBMC在cGMP級培養(yǎng)液CellGro DC中添加GM-CSF和IL-4誘導(dǎo)培養(yǎng)樹突狀細胞(DC),培養(yǎng)過程中添加免疫抑制藥他克莫司獲得tDC。然后采用流式細胞術(shù)檢測他克莫司誘導(dǎo)的tDCCD209、HLA-DR、CD80、CD83、CD86幾種表型分子的表達及其細胞活性。結(jié)果顯示,他克莫司誘導(dǎo)的tDC具有典型的耐受性細胞表型：高表達DC標(biāo)志CD209,低表達共刺激分子CD80、CD86、CD83以及HLA-DR,他克莫司不影響單核細胞向DC的分化。同時,本實驗還采用Annexin V/PI法檢測了他克莫司誘導(dǎo)的tDC的活性,結(jié)果顯示,該法誘導(dǎo)的tDC具有較高的活性。在對他克莫司誘導(dǎo)的tDC的免疫學(xué)功能的研究中,通過CFSE增殖實驗檢測其調(diào)節(jié)異源CD4+效應(yīng)T細胞的作用,同時比較了他克莫司誘導(dǎo)的tDC與Il-10/TGF-β、地塞米松、環(huán)孢素A誘導(dǎo)的tDC的耐受功能。功能分析顯示,與未加藥的相比,藥物地塞米松、環(huán)孢霉素A或他克莫司刺激的tDC具有對異源細胞的低反應(yīng)性,不能誘導(dǎo)異源CD4+T細胞增殖。而他克莫司誘導(dǎo)的tDC更有效地抑制成熟DC引起的混合淋巴細胞反應(yīng)。本實驗也驗證了此方法誘導(dǎo)培養(yǎng)的tDC凍存復(fù)蘇后仍具有表型和功能上的穩(wěn)定性。雖然有研究報道顯示用地塞米松、IL-10、TGF-β等能誘導(dǎo)產(chǎn)生tDC,但對誘導(dǎo)產(chǎn)生的tDC調(diào)節(jié)T細胞的能力很少有研究,本研究建立了他克莫司誘導(dǎo)tDC的培養(yǎng)方法,同時研究了用此法誘導(dǎo)的tDC的免疫學(xué)功能,及凍存復(fù)蘇后的tDC的生物學(xué)特性及功能,證實免疫抑制藥物他克莫司誘導(dǎo)培養(yǎng)的tDC具有耐受功能,為其在自身免疫性疾病、器官移植耐受等方面的治療提供了科學(xué)的實驗室依據(jù),具有重要的現(xiàn)實臨床意義。
[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.

【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R96

【共引文獻】

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