ANALYSIS AND ESTABLISHMENT OF SPECIFIC T-CELL CLONE AGAINST HUMAN TSH-RECEPTOR IN BASEDOW' SDISEASE.

A.人TSHR特异性T细胞的分析：(1)利用聚合酶链式反应(PCR)扩增编码人TSHR胞外区(1.2kb)的人TSHR基因片段，并在Sf9细胞中用Vaculo病毒系统表达相应的蛋白。经SDS-PAGE鉴定为50 kDa，并与抗人TSHR胞外区特异性序列的抗体识别，凝胶渗透层析后，将纯化的蛋白注入兔体内，制备抗体。提高的抗体阻断了TSHR与~(125)I-TSH的结合，表明该抗体是抗TSHR的。(2)模型小鼠TSHR特异性T细胞的分析从Basedow病患者中分离TSHR特异性T细胞克隆尚未成功。因此，首先我们…用脂质体法将人TSHR全长基因导入小鼠成纤维细胞(RT，H-2k单倍型)，获得稳定表达人TSHR的细胞系(RT-TSHR)，免疫Akl/N小鼠(H-2k单倍型)，在脾细胞中形成TSHR。B.抗人TSH受体抗体表位分析：识别TSH受体为ANTO抗原的特异性T细胞和B细胞表位的相关性被认为是非常重要的。因此，我们还研究了针对TSH受体的特异性B细胞表位(即抗体表位)。首先，我们将人促甲状腺激素嵌合体受体部分替换为促性腺激素受体(L H/CGR)。为了明确表位，我们检测了TSH受体刺激性抗体诱导的嵌合体受体中cAMP的积聚。结论：1)刺激性抗体的表位主要位于TSH受体N端的145个氨基酸残基，2)每个表位不同，且依赖于每个刺激性抗体。C.本研究的未来：Basedow病的原因是产生与TSH受体(TSHR)结合并起TSH作用的自身抗体。这是自身免疫病中独特的模型之一。为了阐明该病的发病机制，我们必须弄清参与该病免疫应答的所有因素，特别是通过TSHR的应答。在本研究中，我们在体内诱导了一种针对TSHR的特异性T细胞。通过发展这项技术，我们可以获得一个诱导抗TSHR类Basedow病抗体的模型系统。利用这个检测系统，我们不仅可以确定T细胞或B细胞的表位，而且还可以确定抗原呈递细胞。这是阐明Basedow病发病机制的真正途径。利用嵌合体受体分析抗TSH受体刺激性抗体的表位的工作正在进行中，这些体内模型应该会为认识特异性T细胞和B细胞之间抗TSH受体表位的相关性提供一些建议。然而，刺激性抗体的表位尚未完全阐明，因为识别三维结构的抗体表位与特定T细胞的表位不同，在Basedow病中抗体的表位变化很大。此外，体外和体内模型数据的谨慎比较是最重要的，因为两者总是有好处也有不足。较少

A.Analysis of human TSHR specific T cell : (1) Expression of extracellular domain of human TSHR cDNA fragment encoded extracellular domain of human TSHR (1.2kb) was amplified by polymerase chain reaction (PCR) and the protein correspond to the sequence was expressed in Sf9 cells using vaculo virus system. The protein was migrated as 50kDa by SDS PAGE and recognized with an antibody against specific sequence of extracellular domain of human TSHR.After gel permeation chromatography, the purified protein was injected to rabbits to raise an antibody. The raised antibody blocked binding between TSHR and 125I-TSH to show that the antibody was against for TSHR.From experiments described above, it was clear that the extracellular domain of TSHR expressed in Sf9 cell was suitable for antigen to raise for an antibody. (2) Analysis of TSHR specific T cell in model mouseIt has not been successful to isolate the specific T cell clone for TSHR from patients of Basedow's disease. Therefore, first we … More tried to immunize mouse with TSHR.Human TSHR cDNA (full length) was transfected to mouse fibroblast (RT,H-2k haplotype) with lipofectin to get cell line (RT-TSHR) stably expressed human TSHR.After immunizing RT-TSHR to Akl/N mouse (H-2k haplotype) , we found blast formation for TSHR in spleen cells. It was concluded that, at least, k haplotype in H-2 (mouse MHC) was able to present TSHR molecule to T cell as an antigen.B.Analysis of epitopes for the antibodies against human TSH-receptor : The correlation of the epitopes between the specific T-cell and B-cell, which recognize TSH-receptor as an anto-antigen, is thought to be very important. Therefore, we also studied the epitopes for specific B-cell against TSH-receptor (namely, the epitope for the antibody). At first, we made the chimera-receptors of human TSH partly substituted with leutenizing hormone-receptor (LH/CGR). In order to clarify the epitopes, we checked the accumulation of cAMP in the chimera-receptors induced by the stimulating antibody of TSH-receptor. In conclusions, those were clarified that 1) the epitopes of the stimulating antibody were mainly located within the 145 amino acids residues of N-terminal of TSH-receptor and, 2) the each epitope was different and dependent on the each stimulating antibody.C.Future of this study : The cause of Basedow' disease is the production of autoantibodies that bind the TSH receptor (TSHR) and act as TSH.This is one of the unique model in autoimmnodisease. To elucidate the pothogenesis of this disease, we have to make clear all factors which participate in the immune-response of this disease, especially response through the TSHR.In this study, we induced a specific T-cell against TSHR in vivo. By developping this techniqe, we could get a model system which induce antibodies against TSHR like Basedow' disease. Using this excerent system, we could clarify not only epitopes of T-cell or B-cell but also antigen-presentating cells. This is the real pathway to elucidatethe pothogenesis of Basedow' disease. The analysis of epitopes for the stimulating antibodies against TSH-receptor using the chimera-receptors has been on the way to clarify and these in vivo models should give some suggestions to recognize the correlation of the epitopes between the specific T-cell and B-cell against TSH-receptor. However, the epitopes for the stimulating antibody have not been completely clarified yet because the epitope for the antibody, which recognize the architecture of three-dimentions, is different form the epitope for the specific T-cell, the epitopes of the antibody so varied in the Basedow's diseases. Moreover, the cautions comparison of the data between in vitro and in vivo model is most important since both always have benefit and deficiet. Less