负载滋养层细胞抗原的受体半成熟树突状细胞过继回输致免疫耐受的实验研究

发布时间：2018-02-26 00:08

本文关键词：树突状细胞 Toll样受体原代培养混合淋巴细胞反应滋养层细胞原代培养树突状细胞抗原负载 Th1/Th2反应心脏移植模型心脏移植免疫耐受 T调节细胞 Th1/Th2平衡　出处：《华中科技大学》2007年博士论文　论文类型：学位论文

【摘要】： 树突状细胞(dendritic cell,DC)作为免疫反应的启动者,具有免疫调节的双重性。在移植免疫中,既可诱导产生急、慢性排斥反应,也可在一定条件下,诱导受者外周免疫耐受的建立。移植中DC过继免疫治疗就是体外制备耐受性DC,回输到受者体内,发挥其免疫调节作用,减弱、延迟排斥反应的发生,是极有希望替代免疫抑制治疗的一种诱导免疫耐受的方法。本课题通过体外培养扩增获得耐受性半成熟表型DC,负载滋养层细胞抗原使其获得针对供体的特异性,通过体内、体外实验研究其致免疫耐受的特性和功效,并初步探讨其作用机制,为器官移植中DC过继免疫治疗提供一些新的策略。全文共三个部分。第一部分小鼠髓源性半成熟DC的体外培养和鉴定目的:体外诱导小鼠骨髓细胞分化、增殖为半成熟表型DC,并检测其免疫学功能。方法:使用重组小鼠粒细胞巨噬细胞集落刺激因子(granulocyte macrophage colony stimulating factor, GM-CSF)体外诱导骨髓DC前体细胞分化为未成熟DC,针对Toll样受体信号通路关键蛋白MyD88(myeloid differentiation factor 88)化学合成siRNA抑制DC成熟,同时给予大剂量的脂多糖(lipopolysaccharide ,LPS)刺激得到半成熟表型DC,使用流式细胞术分析DC表面共刺激分子和MHC-II分子的表达,ELISA法检测DC分泌IL-12、IL-10,初次和再次混合淋巴细胞反应(mixed lymphocyte reaction,MLR)检测DC刺激同种淋巴细胞增殖能力。结果:GM-CSF成功诱导骨髓DC前体细胞分化为未成熟DC,MyD88 siRNA可有效抑制DC成熟,体外给予的大剂量LPS刺激可使未成熟DC共刺激分子CD40和MHC-II分子表达上调,分泌IL-10/IL-12比率增高,并分化为半成熟DC。初次MLR中未成熟和半成熟DC均不能有效刺激同种淋巴细胞增殖,再次MLR中半成熟DC较未成熟DC引起同种T细胞更强的免疫无反应性。结论:体外获得的半成熟DC是一种耐受性DC。第二部分负载滋养层细胞抗原对半成熟DC免疫功能的影响实验1滋养层细胞体外培养和鉴定目的:体外培养得到小鼠胎盘滋养层细胞,鉴定其特性和纯度。方法:体外获得孕期小鼠外胎盘锥组织,组织块培养法获得滋养层细胞,使用免疫组化染色和流式细胞术鉴定其特性和纯度。结果:外胎盘锥组织块培养法得到足量、均一的滋养层细胞,PLP-B免疫组化染色阳性率90%,流式细胞术检测MHC-I阳性率为2.16%,证实收获细胞为高纯度的滋养层细胞。结论:外胎盘锥组织块培养法得到的滋养层细胞数量和纯度满意,符合后续实验要求。实验2负载滋养层细胞抗原的半成熟DC免疫学功能研究目的:研究体外负载滋养层细胞抗原对半成熟DC免疫学功能的影响。方法:细胞反复冻融法制备脾细胞抗原和滋养层细胞抗原,体外分别负载半成熟DC,比较DC表型及分泌细胞因子的变化,MLR检测负载抗原后DC致同种淋巴细胞增殖的能力。结果:半成熟DC负载脾细胞抗原后,MHC-II和共刺激分子CD86表达明显增高(P0.05),CD40、CD80无明显变化,分泌IL-12轻度增高,但无统计学意义,体外不能有效刺激同种淋巴细胞增殖;负载滋养层细胞抗原后,DC共刺激分子CD40、CD80、CD86表达均无明显变化,MHC-II表达增高(P0.05),分泌IL-10/IL-12比率增加(P0.05),MLR反应上清液中细胞因子呈现明显Th2优势,不能有效刺激同种淋巴细胞增殖。结论:半成熟DC负载滋养层细胞抗原后,共刺激分子表达无明显变化,自分泌细胞因子呈现更明显的Th2优势,可诱导同种T淋巴细胞特异性无反应性,并优先分化为Th2细胞。第三部分负载滋养层细胞抗原的受体半成熟DC回输对移植心存活时间的影响实验1颈部异位心脏移植模型的改进目的:建立一种难度适中,吻合口长期通畅率高的小鼠颈部异位心脏移植模型的方法。方法:使用Hybrid法建立异位心脏移植模型,将受体鼠右颈总动脉和颈外静脉分别与移植心的无名动脉和肺动脉吻合,其中动脉采用缝合法间断端端缝合,静脉采用套管法端端套扎吻合,和传统缝合法和全套管法比较手术即时效果和术后移植心存活时间。结果:Hybrid法手术成功率高,难度和手术时间界于全套管法和传统缝合法之间,但长期通畅率明显高于全套管法,和传统缝合法无显著性差异,术后移植心存活时间三组无显著性差异。结论:Hybrid法是一种较合理的小鼠颈部异位心脏移植模型的方法。实验2术前回输负载滋养层细胞抗原的受体半成熟DC对移植心存活时间的影响目的:研究负载滋养层细胞抗原的半成熟DC术前回输受体后对移植心存活时间的影响。方法:术前经受体鼠尾静脉回输负载滋养层细胞抗原和负载脾细胞抗原的受体半成熟DC,7天后行颈部同种异位心脏移植,观察移植心存活时间,RT-PCR法检测移植心和受体鼠脾脏细胞细胞因子mRNA表达,流式细胞术检测受体鼠脾脏CD4+CD25+调节性T细胞含量,再次MLR检测受体同种T细胞增殖能力。结果:和负载脾细胞抗原的受体半成熟DC比较,负载滋养层细胞抗原的受体半成熟DC回输更能明显延长移植心存活时间(P0.05),受体鼠T细胞和移植心局部表达IL-10mRNA增高(P0.05),表达IL-2mRNA、IFN-γmRNA降低(P0.05),流式细胞术证实受体脾脏内CD4+CD25+T细胞含量明显增加,MLR显示受体脾脏T细胞对供鼠脾细胞特异性免疫无反应性。结论:负载滋养层细胞抗原的半成熟DC术前回输可诱导受体鼠产生免疫耐受并有效延长移植心存活时间。
[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.

【学位授予单位】：华中科技大学
【学位级别】：博士
【学位授予年份】：2007
【分类号】：R392

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