重症肌無力病人的T細胞接收器α/δ鏈基因使用情形之研究

Abstract

重症肌無力（Myasthenia Gravis, MG）是一種自體免疫疾病，導致病狀的主因是由於體內產生乙醯膽鹼受體專一的自我抗體（autoantibody），阻礙神經肌肉間的訊息傳導，最後造成肌肉無力現象。由實驗動物模型（EAMG）的研究發現必須在有CD4+T細胞存在的情況下，實驗動物才會產生MG症狀，而在人類病患，大部分病人同時伴隨有胸腺（T細胞發育之處）異常症狀（胸腺瘤或胸腺增殖）。因此，T細胞與MG的產生必然有一定的關係。雖然如此，但重症肌無力真正的致病原因仍是待解之謎。 在T細胞接受器（TCR）上V-J接交處的CDR3區域，一向是被認為與T細胞所辨識的抗原性質有關。本論文中，我們取得7位重症肌無力病患與3位對照者的周邊血液淋巴細胞（PBL）。合成對Vα/Vδ、Cα/Cδ基因序列專一之引子，利用RT-PCR/PCRs技術，分析他們α/δ-chain上V基因的使用情形；並進一步對其中的3個患者與1個對照者，做TCR上CDR3區域的DNA定序分析。 首先，我們觀察到在TCR αchain上，Vα14、Vα15這兩個V基因在Jα基因的使用上，似乎有著完全不同的選擇性，且這種使用上的偏好似乎與個人HLA的haplotype無關。 另一方面，我們在兩位病人的樣本中，Vδ2-Jδ-Cδ的基因組合，觀察到一段相似的胺機酸motif；雖然他們所使用的Jδ基因並不相同，但是在VDJ junction部分的序列幾乎完全相同（只有一個胺機酸不同，但性質相似），極有可能辨識的是性質相近的抗原片段，且可能與重症肌無力有關。而這樣的結果則似乎暗示著找到導致重症肌無力相關T細胞的可能性。

Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibody against acetylcholine receptor (AChR). Autoantibody blocked the signal transduction between neuromuscular junction and resulted in muscle weakness. Previously studies in MG’s animal model, experimental autoimmune myasthenia gravis (EAMG), showed that CD4+ T cells are necessary for the production of antibody to AChR. In addition, most MG patients appear to have hyperplasia or thymoma in the thymus, in which T cell development occurs. Thus, it suggests a relationship between T cells and MG formation. However, the real mechanism in causing MG is still unclear. CDR3, the V-J segment junctional region of T cell receptor (TCR), was regarded as the sites contacting directly with antigen presented by MHC molecules. In this study, cDNA of peripheral blood lymphocytes (PBLs) isolated from 7MG patients and 3 healthy controls (HCs) were constructed and analyzed by PCR using V/C sequence-specific primers for TCR Vα/Vδ gene usage. Furthermore, we subcloned and sequenced the PCR products obtained from 3 patients and 1 HC to determine the nucleotide sequences of CDR3 in detail. We observed two interested features. First, the spectrum of Jα gene usage with Vα14 is very different from that with Vα15, suggesting HLA haplotype may not play a role in determining J a usage in conjunction with these two Vαs. Second, we found a similar amino acids motif between two δ chain clones isolated from two different patients. Although two different Jδ genes were used for the two clones, the amino acids sequence of VDJ junctional region are almost identical. Thus, these two clones might recognize similar antigens, and could be relevant with MG. This result suggests the possibility in finding myasthenogenic T cells and may provide an opportunity in immunotreatment for MG.