重症肌無力病人的T細胞接受器基因表現之研究

Abstract

免疫系統是人類及其他脊椎動物用來保護自體免受外來物入侵的一套複雜的生命網絡。 T 細胞所調節的反應在免疫系統中是十分重要的一環。常見的自體免疫疾病如多發性硬化症（ multiple sclerosis , MS ）、類風濕性關節炎（rheumatoid arthritis , R A ）、胰島素依賴型糖尿病（ insulin-dependent diabetes mellitus , IDDM ）等，皆是由於 T 細胞失去對自體抗原的耐受性 ( self - tolerance）而有所反應所造成的，而本論文所研究的對象重症肌無力（myasthenia gravis）則是一種抗體調節（antibody-mediated）的自體免疫疾病，主要的致病原因之一是病人體內產生一種乙醯膽鹼受體（ acetylcholine receptor ）的抗體，造成乙醯膽鹼受體的減少，從重症肌無力的動物模型 EAMG ( experimental autoimmune myasthenia gravis ）與從人類血液中篩選出乙醯膽鹼受體專一的 T 細胞發現， CD4+的輔助 T 細胞（ T helper cells , Th ) 扮演著關鍵的角色。本論文主旨在於分析重症肌無力病人的 T 細胞接受器 ( T cell receptor , TCR ）的基因使用以及 T 細胞接受器 CDR3 部分的核酸序列，希望能藉由這些實驗能分析出與重症肌無力相關的特殊 T 細胞接受器，針對 T 細胞抗原接受器發展出可行的治療方式，將可以更有效的治療重症肌無力。 從本論文分析的 T 細胞接受器的核酸序列中，發現數個出現頻率頗高的特殊使用情形， Vδ2－Ja53－Ca 、Vδ5－Ja49－Ca 、 Vαl4－Jδ1－Cδ，很可能與重症肌無力的致病原因相關，值得進一步的探討。 此外，還有一些 T 細胞接受器 CDR3 的序列與現存的基因庫比對極為相近相似甚至完全相同，它們所對應的 T 細胞都有其特殊的性質，而它們是否與重症肌無力有關，也需要更進一步的實驗才能證明。 因此我們希望根據目前所得到的結果，繼續針對特殊表現的 T 細胞接受器做進一步的研究，找出重症肌無力特殊的 T 細胞。

Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibodies against acetylcholine receptor (AChR). T lymphocytes in MG patients are considered to exert a regulatory function in the development of antiAchR antibodies. These autoreactive T cells may be activated through recognition of self-antigen, such as AchR and/or AchR-related molecules and complementarity determining region 3 (CDR3) in the V region of T cell receptor (TCR) molecules is regarded as the major site responsible for the antigen-recognition. In this study, we analyzed in detail the TCR Vα and Vδ gene usage in peripheral blood lymphocytes (PBL) of a MG patient as an initial step in understanding the relevancy of TCRs in the pathogenic T cells. Several Vα/Vδ and Cα/Cδ sequence-specific primers were synthesized and used in RT-PCR/PCRs to obtain TCR α/δ-chain genes present in PBLs of the MG patient. PBLs from the patient’s brother who has no MG symptoms were also analyzed in parallel to distinguish the effect caused by either MHC alone or MHC plus the potential pathogenic antigens on the observed TCR V gene usage Dominant usage of certain TCR V—J combination, that is Vδ2-Jaα53-Cα, Vδ5-Jα49-Cα and Vα1 4-Jδ1 -Cδ, is observed in the samples of the MG patient, but not in the isolates from patient’s brother. Moreover, these TCR genes were suggested to be isolated from oligoclonally-expanded T cells since identical V-J junctional sequences were evidenced by nucleotide sequence analysis. It, therefore, indicates that the T cells bearing these TCRs may be activated by certain antigens (could be AchR or AchR-like peptides) in the MG patient and suggests that these T cells might play an important role in generating MG. In addition, some CDR3 sequences in the TCRs isolated from the MG patient are identical or homologous to the CDR3 sequences in TCRs of some previously identified T cells, such as DNαβ and γδ cells. The functional significance of these TCR genes present in the MG patient is discussed in this report.