γδT细胞在皮肤移植免疫排斥反应中的作用及机理初探

发布时间：2018-03-18 13:18

本文选题：γδT细胞　切入点：DETC　出处：《第三军医大学》2015年硕士论文　论文类型：学位论文

【摘要】：研究背景异体皮肤移植以后引发的强烈免疫排斥反应是阻碍大面积烧伤患者创面修复的关键问题。经典免疫学观点认为,LCs是在异体皮肤移植免疫排斥反应中起核心作用的免疫细胞。但通过LCs敲除小鼠皮肤移植模型发现,与供受体都是野生型小鼠的皮肤移植相比,供体、受体以及供受体都缺乏LCs的小鼠之间的皮肤移植,移植物存活时间并没有显著延长。提示在皮肤移植免疫排斥反应中还存在其它细胞参与。而另外一种重要的构成皮肤组织免疫微环境的细胞是γδT细胞,其在肿瘤、感染、创面愈合、自身免疫性疾病等发挥重要作用。我们前期预实验也通过小鼠皮肤移植模型发现,对比野生型小鼠的皮肤移植物,TCRδ基因敲除小鼠的皮肤移植物存活时间明显延长,提示γδT细胞可能在异体皮肤移植免疫排斥反应中起重要作用,且γδT细胞是皮肤组织中分泌细胞因子的重要来源。而可能参与皮肤排斥反应的有着不同功能的γδT细胞亚群包括:1.分布于表皮组织中的Vγ3 T细胞即树突状表皮T细胞(Dendritic Epidermal T Cells,DETCs);2.分布于真皮组织中的Vγ4 T细胞;3.分布于外周循环的Vγ4 T细胞。究竟是谁在皮肤移植免疫排斥反应中起重要作用、如何发挥作用以及具体作用机制迄今尚不清楚,阐明这些问题,不仅能够使我们进一步了解皮肤移植免疫排斥反应的特殊性,而且可以作为传统移植排斥反应的有益补充,并为临床控制皮肤免疫排斥反应提供新的细胞分子靶点。目的本研究拟探讨γδT各亚群是否参与异体皮肤移植免疫排斥反应以及相应机制。方法1、通过建立同系MHC分子微小差异的不同性别小鼠(雄-雌)之间的皮肤移植模型,比较野生型组、TCRδ-/-组移植皮片存活时间。2、通过免疫荧光的方法观察DETCs的一般形态;流式细胞技术分析DETCs的表型及细胞因子的表达情况;通过将DETCs和na?ve CD4+T细胞体外进行共培养,观察DETCs对na?ve CD4+T细胞分化方向的影响。3、通过建立小鼠皮肤移植模型,比较野生型组和Vγ4清除组移植皮片存活时间。并在移植后第5天,取野生型组移植皮片分离表皮细胞作为实验组,同体正常表皮作为对照组,流式细胞术分析Vγ4 T细胞的浸润情况,Western blot检测移植皮片表皮组织中IL-17、IL-23、IL1β的表达情况。结果1、TCRδ-/-组皮肤移植物存活时间较野生型组皮肤移植物存活时间显著延长。皮肤移植物平均生存时间(MST)(TCRδ-/-组vs野生型组:22.30天vs 10.60天P0.001)。2、共聚焦显微镜下观察DETCs的形态,呈APC的树突状细胞的特征,活化后分泌一定水平的IFN-γ和IL-17A,体外与na?ve CD4+T细胞进行共培养可以诱导后者向Th1方向分化。3、Vγ4清除组皮肤移植物较野生型组皮肤移植物存活时间显著延长。皮肤移植物平均生存时间(MST)(TCRδ-/-组vs野生型组:49.08天vs 11.00天P0.001)。4、野生型小鼠移植后第5天,皮肤移植物表皮组织较正常表皮浸润Vγ4 T细胞数量明显增加,由0.86%增加到6.08%,而DETCs比例显著下降,由2.38%下降到0.78%。移植物表皮组织中IL-17、IL-23、IL1β的表达较正常表皮显著增强(P0.05)。结论本研究初步证实γδT细胞参与并加速了皮肤移植排斥反应的发生发展过程,而不同亚型的γδT细胞在其中发挥的作用有所不同。Vγ3γδT细胞(即DETCs)具有抗原递呈细胞的基本特征,可能是在异体皮肤免疫排斥反应中起关键作用的DCs;而Vγ4γδT细胞通过早期提供IL-17来加速皮肤移植排斥反应的发生。综上所述,γδT细胞作为皮肤免疫微环境的重要组成部分,并在皮肤移植免疫排斥反应中具有其特殊的作用。
[Abstract]:On the background of strong immune skin allograft rejection is caused after the key problems that hinder the repair of large area burn wound. The classical immunology point of view, LCs is in the immune skin allograft rejection plays a central role in the reaction of immune cells. But through the LCs knockout mouse model of skin transplantation, and for all the wild type receptor mice skin transplantation compared to donor, receptor and receptor for lack of LCs mice skin transplantation, graft survival was not significantly prolonged. In skin allograft rejection also exist in other cells. Another important constituent of skin tissue cells are the immune microenvironment of gamma delta T cells in the tumor, infection, wound healing, an important role in autoimmune disease. We play a pre experiment through mouse skin transplantation model and found that compared to the wild Type of mouse skin graft, TCR delta knockout mouse skin allograft survival time was significantly prolonged, suggesting that T cells may have immune rejection in allograft skin plays an important role in the reaction, and gamma delta T cells is an important source of cytokine secretion in skin tissue and may be involved in the rejection of skin there are different functions of gamma delta T cell subsets including V gamma 1. distributed in the epidermal tissue 3 T cells called dendritic epidermal T cells (Dendritic Epidermal T Cells, DETCs); V gamma 2. distributed in the dermal tissues of 4 T cells in the peripheral circulation; 3. V gamma ring 4 T who is in the cell. The skin graft rejection reaction plays an important role in how to play the role and the specific mechanism remains unclear. To clarify these issues, not only enables us to further understand the particularity of skin allograft rejection, and can be used as A useful complement to traditional transplant rejection, and reaction cell new molecular targets for the clinical control of skin immune rejection. The purpose of this study is to explore the gamma delta T subsets in immune skin allograft rejection and corresponding mechanism. 1, through different sex mice homologous MHC molecules to small differences (male - female) skin transplantation model between the wild type group, TCR 8 - group of skin graft survival time.2, we observed the morphology of DETCs by immunofluorescence; analysis of phenotype and cytokine DETCs flow cytometry the expression of DETCs and Na; the CD4+T cells were co cultured with ve? In vitro, the effect of DETCs on the na?.3 ve CD4+T influence cell differentiation direction, through the establishment of a mouse model of skin transplantation, compared with wild type group and V gamma 4 clearance group skin graft survival time. And in fifth days after transplantation, the wild type group Skin grafts isolated epidermal cells as the experimental group, normal skin as control group, the infiltration of flow cytometric analysis of V gamma 4 T cells, IL-17, Western blot detection of skin grafts in the epidermis of IL-23, expression of IL1 beta. 1, TCR 8 - group of skin allograft survival compared to the wild type of skin graft survival time was significantly prolonged. The average survival time of skin graft (MST) (TCR Delta - group vs wild type group: 22.30 days vs 10.60 days P0.001.2), to observe the morphology of DETCs confocal microscope, a feature of APC dendritic cells, secretion of a certain level of activation of IFN- and gamma IL-17A, Na and VE in vitro? CD4+T cells can induce the differentiation towards the direction of Th1.3 co cultured with V gamma 4 clearance group skin graft compared with the wild type group of skin graft survival time was significantly prolonged. The average survival time of skin graft (MST) - vs (TCR delta group of wild type Group: 49.08 days vs 11 days P0.001.4), wild type mice fifth days after transplantation, skin graft of epidermal tissue than normal epidermal infiltration of V gamma 4 the number of T cells increased significantly, increased from 0.86% to 6.08%, while the proportion of DETCs was significantly decreased, decreased from 2.38% to IL-17, 0.78%. graft in the epidermis of IL-23 expression IL1 beta than normal epidermis increased significantly (P0.05). Conclusion: T cells participate in and accelerate the process of the occurrence and development of skin allograft rejection, and different subtypes of gamma delta T cells which play the role of different.V Gamma 3 gamma delta T cells (DETCs) with antigen is the basic feature of cell, may be in the allograft immune rejection plays a key role in the reaction of DCs; and 4 V gamma gamma delta T cells by early IL-17 to accelerate the occurrence of skin allograft rejection. In conclusion, an important group of gamma delta T cells as the skin immune micro environment In part, it has a special role in the immune rejection of skin transplantation.

【学位授予单位】：第三军医大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R622.1

【相似文献】