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

巴氏疾病中针对人类 TSH 受体的特异性 T 细胞克隆的分析和建立。

基本信息

批准号：
05670839
负责人：
TAHARA Kazuo
金额：
$ 1.34万
依托单位：
CHIBA UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for General Scientific Research (C)
财政年份：
1993
资助国家：
日本
起止时间：
1993 至 1994
项目状态：
已结题

项目摘要

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

A.人TSHR特异性T细胞的分析：（1）人TSHR胞外区的表达通过PCR扩增人TSHR胞外区cDNA片段（1.2kb），并利用空泡病毒系统在Sf 9细胞中表达相应的蛋白。经SDS PAGE电泳，该蛋白分子量为50 kDa，与抗人TSHR胞外区特异性序列的抗体结合，经凝胶渗透色谱纯化后，注射家兔体内，制备抗体。所产生的抗体阻断了TSHR与125 I-TSH的结合，表明该抗体是抗TSHR的，从上述实验可以清楚地看出，在Sf 9细胞中表达的TSHR的胞外区适合于抗原以产生抗体。(2)模型小鼠TSHR特异性T细胞的分析从巴塞杜病患者中分离TSHR特异性T细胞克隆尚不成功。因此，我们首先 ...更多信息将人TSHR cDNA（全长）经脂质体转染小鼠成纤维细胞（RT，H-2k单倍型），获得稳定表达人TSHR的细胞株（RT-TSHR），将RT-TSHR免疫Akl/N小鼠（H-2k单倍型）后，在脾细胞中观察到TSHR原始细胞的形成。结论是，至少H-2（小鼠MHC）中的k单倍型能够将TSHR分子作为抗原呈递给T细胞.B.抗人TSH受体抗体的表位分析：识别TSH受体作为自身抗原的特异性T细胞和B细胞之间的表位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细胞的表位（即抗体的表位）。首先，我们用促黄体生成素受体（LH/CGR）部分取代人TSH嵌合受体。为了阐明表位，我们检测了TSH受体刺激抗体诱导的嵌合受体中cAMP的积累。综上所述，阐明了1）刺激性抗体的表位主要位于TSH受体N端145个氨基酸残基内，2）每个表位是不同的，并依赖于每个刺激性抗体。C.本研究的未来：Basedow病的病因是自身抗体的产生，这些抗体结合TSH受体（TSHR）并充当TSH，这是自身免疫病的独特模型之一。为了阐明本病的发病机制，我们必须弄清参与本病免疫应答的所有因素，特别是通过TSHR的免疫应答。通过该技术的发展，我们可以建立一个类似巴塞多病的抗TSHR抗体的模型系统。利用该系统，不仅可以明确T细胞或B细胞的抗原表位，而且可以明确抗原提呈细胞。这是阐明巴塞杜病发病机制的真实的途径。利用嵌合受体对促甲状腺激素受体抗体表位的分析已逐渐明确，这些体内模型为认识促甲状腺激素受体特异性T细胞和B细胞之间表位的相关性提供了一些建议。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的表位不同于特异性T细胞的表位，抗体的表位在Basedow病中变化如此之大。此外，体外和体内模型之间的数据的谨慎比较是最重要的，因为两者总是有利有弊。少