【作者】 肇静娴；

【导师】 曾耀英；

【作者基本信息】 暨南大学 ， 病理生理学， 2001， 硕士

【摘要】 目的：本研究旨在建立一个快速、准确的同种异基因抗原识别的评价系统，以便对同种移植排斥机理进行深入的研究。 方法：本研究所使用的移植抗原识别模型如下：以混合淋巴细胞培养为体外同种异基因抗原识别的研究模型，以心肌组织耳廓移植为移植局部观察模型，以心肌组织前臂皮下移植为同种异型抗原间接提呈模型，以分离单个肝细胞腹腔移植为同种异型抗原直接和间接提呈模型。主要的评价指标为T细胞活化CD69等表面分子和细胞因子的表达。用荧光标记的单克隆抗体对T细胞表面分子或细胞内细胞因子进行双色或三色免疫荧光标记，用流式细胞仪进行分析。 结果：在混合淋巴细胞培养实验系统里，应答细胞培养48小时CD69和CD25表达百分率均明显高于对照组，分别为20.65±1.0对7.16±0.21和40.54±1.87对16.91±0.93；应答T细胞IFN-γ等细胞因子的表 同种异基困抗原识别评价系统的研究 中荚文摘要达百分率也明显高于对照组，其值为18．65 LI．00对1．Zo t 0．16。与同基困移植情况相比，耳廓同种异基因心肌组织移植局部免疫炎症、血管扩张明显可见。与同基因移植情况相比，前臂同种异基卧C肌组织移植48，］、时后腋窝淋巴结m41细胞和 CDS1细胞表达CD69均见明显升高。在移植的同时局部给予弗氏完全佐剂可强化同种异基固抗原刺激T细胞表达CD69。腹腔同种异基因肝细胞移植可导致肠系膜淋巴结 T细胞CD69表达增加，而环胞素A可抑制其表达。用17个针对nR刁叩基因不同片段编码蛋白的单克隆抗体分析nR斗叩基因谱，未发现腹腔同种异基因肝细胞移植导致肠系膜淋巴结 T细胞的 TCR－Vg基困片段谱出现选择性取用情况。 结论：研究结果表明，我们已建立了一个新的MLC方法。与传统的MLC相比，我们的 MLC方法有以下几个方面的优点：不必使用同位素，耗时减少，不必对刺激细胞进行照射等预处理，在单个细胞水平上反映同种异型抗原的识别，能在一定的程度上反映同种异型抗原刺激所引发的免疫应答的模式，而且具有了解不同T细胞亚群同种异型抗原识别和不同洲C等位基因产物配对识别的潜能。耳廓。G肌移植为很好的研究移植局部免疫应答的模型。前臂组织移植和腹腔细胞移植并观察引流淋巴结T细胞活化对评价药物及其它免疫干预作用是十分有用的模型。更多还原

【Abstract】 Aim: The purpose of this study is to establish a fast and precision evaluating system on alto-recognition for further study on mechanism by which allograft is rejected. Methods: The models for transplant antigen recognition used in this study are as follow. Mixed lymphocyte culture (MLC) is used as the model for studying in vitro alto-recognition. Transplant of myocardio-tissue into mouse ear subcutaneously is used as a model for local observation of graft. Transplant of myocardio-tissue into mouse forearm subcutaneously is used as a model for investigation of direct recognition of alto-antigen. And, abdomino-transplant of isolated hepatocytes is used as a model for investigation of both direct and indirect recognition of alto-antigen. The main parameters include expression of surface molecules (for example CD69) and cytokines during T cell activation. The T cell surface molecules and intracellular cytokines were marked with fluorescence conjugated antibodies, and detected by flow cytometry. Results: In the mixed lymphocyte culture system, the percentages of CD69 and CD25 by the responder cells during 48 hour culture were significantly higher than those from the control group, respectively 20.65 .0 vs.7.16 .2I,40.54 1.87 vs.16.91 .93. While the percentages of IFN-y by the responder T cells were significantly higher than those from the control group, respectively 18.65 .00 vs. 1.20 0.16. As compared with isograft, there is more significant evidence of immunological inflammation and vascular dilatation in the local area in the mouse with myocardio-tissue graft in ear. Compared to isograft, the expression rates of CD69 by the CD4 T cell and CD8 T cell from the drainage lymphnode in the mouse with allo-myocardio-graft were significantly higher. Treatment with Freund’s complete adjuvant during transplantation enhanced CD69 expression by the T cells stimulated by alto-antigens. Abdomino-transplant of isolated hepatocytes resulted in an increase of expression of CD69 by T cells from the mesenteric lymph nodes, which could be inhibited by cyclosporin A. No selective usage of TCR Vf3 segment was found in T cell population from the mcsenteric lymph nodes of the mouse with abdomino-transplant of isolated allo-hepatocytes when the detection was performed with 17 anti-TCR VJ3 antibodies. Conclusion: Our results showed that we have created a new method of MLC. Compared to the traditional method, our method has the advantage of no isotope need, time saving, no pretreatment (irradiation) of stimulator cells, understanding of alto-recognition in single cell level, reflection of the mode of alto-reaction and capability of understanding of alto-recognition by different T cell subsets and combination of different MHC alleles. Myocardio-tissue transplant into mouse ear is a good model for local observation of immune response. Tissue transplant into forearm or abdominal cavity and analysis of activation of T cell from the drainage lymph node was an useful model for evaluating effect of drugs or other immune interventional factors.更多还原