本文關(guān)鍵詞： 樹突狀細胞 Toll樣受體 原代培養(yǎng) 混合淋巴細胞反應(yīng) 滋養(yǎng)層細胞 原代培養(yǎng) 樹突狀細胞 抗原負載 Th1/Th2反應(yīng) 心臟移植 模型 心臟移植 免疫耐受 T調(diào)節(jié)細胞 Th1/Th2平衡　出處：《華中科技大學》2007年博士論文　論文類型：學位論文

【摘要】： 樹突狀細胞(dendritic cell,DC)作為免疫反應(yīng)的啟動者,具有免疫調(diào)節(jié)的雙重性。在移植免疫中,既可誘導產(chǎn)生急、慢性排斥反應(yīng),也可在一定條件下,誘導受者外周免疫耐受的建立。移植中DC過繼免疫治療就是體外制備耐受性DC,回輸?shù)绞苷唧w內(nèi),發(fā)揮其免疫調(diào)節(jié)作用,減弱、延遲排斥反應(yīng)的發(fā)生,是極有希望替代免疫抑制治療的一種誘導免疫耐受的方法。本課題通過體外培養(yǎng)擴增獲得耐受性半成熟表型DC,負載滋養(yǎng)層細胞抗原使其獲得針對供體的特異性,通過體內(nèi)、體外實驗研究其致免疫耐受的特性和功效,并初步探討其作用機制,為器官移植中DC過繼免疫治療提供一些新的策略。全文共三個部分。 第一部分小鼠髓源性半成熟DC的體外培養(yǎng)和鑒定 目的:體外誘導小鼠骨髓細胞分化、增殖為半成熟表型DC,并檢測其免疫學功能。 方法:使用重組小鼠粒細胞巨噬細胞集落刺激因子(granulocyte macrophage colony stimulating factor, GM-CSF)體外誘導骨髓DC前體細胞分化為未成熟DC,針對Toll樣受體信號通路關(guān)鍵蛋白MyD88(myeloid differentiation factor 88)化學合成siRNA抑制DC成熟,同時給予大劑量的脂多糖(lipopolysaccharide ,LPS)刺激得到半成熟表型DC,使用流式細胞術(shù)分析DC表面共刺激分子和MHC-II分子的表達,ELISA法檢測DC分泌IL-12、IL-10,初次和再次混合淋巴細胞反應(yīng)(mixed lymphocyte reaction,MLR)檢測DC刺激同種淋巴細胞增殖能力。 結(jié)果:GM-CSF成功誘導骨髓DC前體細胞分化為未成熟DC,MyD88 siRNA可有效抑制DC成熟,體外給予的大劑量LPS刺激可使未成熟DC共刺激分子CD40和MHC-II分子表達上調(diào),分泌IL-10/IL-12比率增高,并分化為半成熟DC。初次MLR中未成熟和半成熟DC均不能有效刺激同種淋巴細胞增殖,再次MLR中半成熟DC較未成熟DC引起同種T細胞更強的免疫無反應(yīng)性。 結(jié)論:體外獲得的半成熟DC是一種耐受性DC。 第二部分負載滋養(yǎng)層細胞抗原對半成熟DC免疫功能的影響 實驗1滋養(yǎng)層細胞體外培養(yǎng)和鑒定 目的:體外培養(yǎng)得到小鼠胎盤滋養(yǎng)層細胞,鑒定其特性和純度。 方法:體外獲得孕期小鼠外胎盤錐組織,組織塊培養(yǎng)法獲得滋養(yǎng)層細胞,使用免疫組化染色和流式細胞術(shù)鑒定其特性和純度。 結(jié)果:外胎盤錐組織塊培養(yǎng)法得到足量、均一的滋養(yǎng)層細胞,PLP-B免疫組化染色陽性率90%,流式細胞術(shù)檢測MHC-I陽性率為2.16%,證實收獲細胞為高純度的滋養(yǎng)層細胞。 結(jié)論:外胎盤錐組織塊培養(yǎng)法得到的滋養(yǎng)層細胞數(shù)量和純度滿意,符合后續(xù)實驗要求。 實驗2負載滋養(yǎng)層細胞抗原的半成熟DC免疫學功能研究 目的:研究體外負載滋養(yǎng)層細胞抗原對半成熟DC免疫學功能的影響。 方法:細胞反復(fù)凍融法制備脾細胞抗原和滋養(yǎng)層細胞抗原,體外分別負載半成熟DC,比較DC表型及分泌細胞因子的變化,MLR檢測負載抗原后DC致同種淋巴細胞增殖的能力。 結(jié)果:半成熟DC負載脾細胞抗原后,MHC-II和共刺激分子CD86表達明顯增高(P0.05),CD40、CD80無明顯變化,分泌IL-12輕度增高,但無統(tǒng)計學意義,體外不能有效刺激同種淋巴細胞增殖;負載滋養(yǎng)層細胞抗原后,DC共刺激分子CD40、CD80、CD86表達均無明顯變化,MHC-II表達增高(P0.05),分泌IL-10/IL-12比率增加(P0.05),MLR反應(yīng)上清液中細胞因子呈現(xiàn)明顯Th2優(yōu)勢,不能有效刺激同種淋巴細胞增殖。 結(jié)論:半成熟DC負載滋養(yǎng)層細胞抗原后,共刺激分子表達無明顯變化,自分泌細胞因子呈現(xiàn)更明顯的Th2優(yōu)勢,可誘導同種T淋巴細胞特異性無反應(yīng)性,并優(yōu)先分化為Th2細胞。 第三部分負載滋養(yǎng)層細胞抗原的受體半成熟DC回輸對移植心存活時間的影響 實驗1頸部異位心臟移植模型的改進 目的:建立一種難度適中,吻合口長期通暢率高的小鼠頸部異位心臟移植模型的方法。 方法:使用Hybrid法建立異位心臟移植模型,將受體鼠右頸總動脈和頸外靜脈分別與移植心的無名動脈和肺動脈吻合,其中動脈采用縫合法間斷端端縫合,靜脈采用套管法端端套扎吻合,和傳統(tǒng)縫合法和全套管法比較手術(shù)即時效果和術(shù)后移植心存活時間。 結(jié)果:Hybrid法手術(shù)成功率高,難度和手術(shù)時間界于全套管法和傳統(tǒng)縫合法之間,但長期通暢率明顯高于全套管法,和傳統(tǒng)縫合法無顯著性差異,術(shù)后移植心存活時間三組無顯著性差異。 結(jié)論:Hybrid法是一種較合理的小鼠頸部異位心臟移植模型的方法。 實驗2術(shù)前回輸負載滋養(yǎng)層細胞抗原的受體半成熟DC對移植心存活時間的影響 目的:研究負載滋養(yǎng)層細胞抗原的半成熟DC術(shù)前回輸受體后對移植心存活時間的影響。 方法:術(shù)前經(jīng)受體鼠尾靜脈回輸負載滋養(yǎng)層細胞抗原和負載脾細胞抗原的受體半成熟DC,7天后行頸部同種異位心臟移植,觀察移植心存活時間,RT-PCR法檢測移植心和受體鼠脾臟細胞細胞因子mRNA表達,流式細胞術(shù)檢測受體鼠脾臟CD4+CD25+調(diào)節(jié)性T細胞含量,再次MLR檢測受體同種T細胞增殖能力。 結(jié)果:和負載脾細胞抗原的受體半成熟DC比較,負載滋養(yǎng)層細胞抗原的受體半成熟DC回輸更能明顯延長移植心存活時間(P0.05),受體鼠T細胞和移植心局部表達IL-10mRNA增高(P0.05),表達IL-2mRNA、IFN-γmRNA降低(P0.05),流式細胞術(shù)證實受體脾臟內(nèi)CD4+CD25+T細胞含量明顯增加,MLR顯示受體脾臟T細胞對供鼠脾細胞特異性免疫無反應(yīng)性。 結(jié)論:負載滋養(yǎng)層細胞抗原的半成熟DC術(shù)前回輸可誘導受體鼠產(chǎn)生免疫耐受并有效延長移植心存活時間。
[Abstract]:Dendritic cells (dendritic cell DC) as the initiation of immune reaction, has dual immunomodulatory. In transplantation immunity, can induce acute and chronic rejection, but also under certain conditions, the establishment of peripheral immune tolerance induced by DC. The adoptive immunotherapy is prepared in vitro transplantation the tolerance of DC, returning it to the recipient, to play its role in immune regulation, reduced delay rejection, is a method of inducing immune tolerance to treatment inhibited the immune replacement. The subject was amplified by semi mature DC resistant phenotype in vitro, loaded with trophoblastic cells antigen to obtain according to the specific donor by in vivo in vitro, the immune tolerance induced by the characteristics and efficacy, and to explore its mechanism, to provide some new strategies for organ transplantation in the treatment of adoptive immune DC. There are three Part.
In vitro culture and identification of medullary semi mature DC in part 1 of mice
Objective: to induce the differentiation of mouse bone marrow cells in vitro, and to proliferate to semi mature DC, and to detect its immunological function.
Methods: using recombinant mouse granulocyte macrophage colony stimulating factor (granulocyte macrophage colony stimulating factor, GM-CSF DC) inducing bone marrow progenitor cells differentiate into immature DC in Toll like receptor signaling pathway key protein MyD88 (myeloid differentiation factor 88) chemical synthesis of siRNA inhibits DC maturation, while giving high doses of lipopolysaccharide (lipopolysaccharide, LPS) stimulation by semi mature phenotype of DC, using the expression analysis of costimulatory molecules and MHC-II molecules on DC surface by flow cytometry, DC ELISA detection method IL-12 IL-10, secretion, primary and secondary mixed lymphocyte reaction (mixed lymphocyte reaction, MLR) detection of DC stimulate allogenic lymphocyte proliferation.
Results: GM-CSF induced DC bone marrow progenitor cells differentiate into immature DC, MyD88 siRNA can effectively inhibit DC maturation in vitro given large doses of LPS can stimulate the maturation of DC costimulatory molecules CD40 and MHC-II molecule expression and secretion of IL-10/IL-12 ratio increased, and differentiate into immature DC. MLR in the first half ripe and semi mature DC can not effectively stimulate allogeneic lymphocyte proliferation, MLR DC is again half mature and immature DC induce immune alloreactive T cells more reactive.
Conclusion: the semi mature DC obtained in vitro is a tolerable DC..
The effect of second part loaded trophoblast antigen on the immune function of semi mature DC
In vitro culture and identification of Experiment 1 trophoblast cells
Objective: to obtain the mouse placental trophoblast cells in vitro, and to identify the characteristics and purity of the cells.
Methods: in vitro mouse ectoplacental cone tissue obtained trophoblast tissue culture method, using immunohistochemical staining and flow cytometry analysis of its characteristics and purity.
Results: the ectoplacental cone tissue culture method to obtain enough uniform, trophoblast cells, immunohistochemical staining of PLP-B positive rate was 90%. Flow cytometry was used to detect MHC-I positive rate was 2.16%, the cells were harvested for confirmed high purity of trophoblast cells.
Conclusion: ectoplacental cone cultured trophoblast cells quantity and purity were satisfied, meet the demands of further experiments.
Study of semi mature DC immunological function of Experiment 2 loaded trophoblast cell antigen
Objective: To study the effect of in vitro loaded trophoblast antigen on the immunological function of semi mature DC.
Methods: splenocytes antigens and trophoblast cell antigens were prepared by repeated freezing and thawing. They were loaded with semi mature DC in vitro. The DC phenotype and secretion of cytokines were compared. MLR was used to detect the ability of DC to induce allogeneic lymphocyte proliferation after loading antigen.
Results: the semi mature DC loaded with spleen cell antigen, MHC-II and costimulatory molecule CD86 expression was significantly higher (P0.05), CD40, CD80 had no obvious change, the secretion of IL-12 increased slightly, but without statistical significance. In vitro can not effectively stimulate allogenic lymphocytes proliferation of trophoblast cells; load antigens, DC costimulatory molecules CD40, CD80. The expression of CD86 had no significant changes, increased expression of MHC-II (P0.05), IL-10/IL-12 (P0.05) secretion rate increased, cell factor MLR reaction supernatant showed the advantages of Th2, can not effectively stimulate allogeneic lymphocyte proliferation.
Conclusion: the expression of costimulatory molecules is not changed after semi mature DC loaded trophoblast antigen. Autocrine cytokines show a more obvious Th2 advantage, which can induce specific T cell specific reactivity and preferentially differentiate into Th2 cells.
The effect of semi mature DC back transfusion on the survival time of the third part of the recipient trophoblast cell antigen
Experimental 1 model improvement of ectopic heart transplantation in the neck
Objective: to establish a model of heterotopic heart transplantation in the neck of mice with moderate difficulty and high rate of long term patency of the anastomotic stoma.
Methods: to establish the model of heterotopic heart transplantation by using Hybrid method, the recipient right common carotid artery and external jugular vein respectively and transplantation of heart innominate artery and pulmonary artery anastomosis, the artery by suture intermittent end-to-end suture, vein ligation method using the casing end to end anastomosis, and traditional suture and Benoto method heart survival to compare the surgical effect and transplantation time immediately after surgery.
Results: the success rate of Hybrid operation was high, and the difficulty and operative time were between the full casing method and the traditional suture method. But the long-term patency rate was significantly higher than that of the full casing method. There was no significant difference between the traditional method and the traditional suture method. There was no significant difference in the survival time between the three groups after operation.
Conclusion: the Hybrid method is a more reasonable method for the model of heterotopic heart transplantation in the neck of mice.
Effect of semi matured DC receptor DC on the survival time of transplantation of trophoblast cell antigen
Objective: To study the effect of pre transfused receptor on the survival time of the transplanted heart after the semi matured DC receptor of trophoblast antigen (HLA).
Methods: the preoperative receptor rat tail vein transfusion trophoblastic cells antigen load and load of spleen cell antigen receptor semi mature DC, 7 days after cervical heterotopic heart transplantation, graft survival was observed, RT-PCR method was used to detect the expression of transplanted heart and spleen cell cytokine receptor of rat mRNA receptor, detection of spleen CD4+CD25+ regulatory T cells in flow cytometry, again MLR was used to detect the expression of allogeneic T cell proliferation ability.
Results: compared with the load of spleen cell antigen receptor semi mature DC, loaded with trophoblastic cells antigen receptor DC transfusion semi mature can obviously prolong the survival time of the transplanted heart (P0.05), the receptor of rat T cells and heart transplantation increased local expression of IL-10mRNA (P0.05), the expression of IL-2mRNA, reduce the IFN- mRNA (P0.05), confirmed by gamma CD4+CD25+T cell receptor content in the spleen was significantly increased by flow cytometry, MLR receptor spleen T cell anergy of donor specific immune spleen cells.
Conclusion: pre transfusion of semi mature DC with loaded trophoblast antigen can induce receptor mice to produce immune tolerance and prolong the survival time of the transplanted heart.

【學位授予單位】：華中科技大學
【學位級別】：博士
【學位授予年份】：2007
【分類號】：R392

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相關(guān)期刊論文 前1條

1 馮劍鍔;孫宗全;;應(yīng)用Cuff技術(shù)建立小鼠異位心臟移植模型[J];中華實驗外科雜志;2005年12期

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