- 學位論文
肌無力病人體內致病T細胞接受器α鏈基因的研究
Isolation of Potential Pathogenic TCR ?chain Genes in Myasthenia Gravis Patients
摘要
肌無力症是一種自體免疫疾病，主要的致病原因是由於病人體內產生一種乙醯膽鹼受體（acetylcholine receptor）的抗體，因而阻礙神經的傳導，並造成乙醯膽鹼受體的減少。本論文主旨在分析肌無力病人的T細胞接受器?鴗WVδ5及V?的基因使用，以及這些T細胞接受器CDR3 區域的核酸序列，希望藉由這些實驗能分析出與肌無力症相關的致病的T細胞，以期望可以藉由知道這些T細胞接受器的基因序列發展出專一的治療方式，而能更有效的治療肌無力症。 本論文中，我們取得四位肌無力病人（MG-D, MG-Y, MG-Ⅳ, MG-L）和三位正常人（HC-W, HC-R, HC-A）的周邊血液，及MG-L的胸腺內T淋巴細胞及甲狀腺的組織侵入性淋巴球(tissue infiltrating lymphocyte)來作分析，利用RT-PCR的技術，分析Vδ5-Cㄓ垝?-Cㄢo兩種基因組合在肌無力病人和正常人的表現情形，並進一步做T細胞接受器上CDR3區域的DNA定序分析。 首先，我們觀察到在Vδ5-Cㄟ穧]的使用上，肌無力病人的T細胞接受器CDR3區域的第六個或第七個（junction區域）帶有Arginine（R）正電荷氨基酸的比例比正常人高出很多，這樣的結果和EAMG老鼠所得到的結果有原先未料到的高符合性，因此，我們推斷此類的T細胞應該就是所謂的致病的T細胞。 在V?-Cㄟ穧]的使用上，除了MG-Ⅳ以外，其餘肌無力病人和正常人間T細胞接受器CDR3區域的氨基酸分子並沒有什麼差別，但在V?-Cㄟ穧]的使用上，我們找到了新的，可說是人類第三條的invariant TCR，也就是V?-J?2-Cㄢo條T細胞接受器?魽A而這類T細胞接受器顯然被病人的CD4+CD8+DP T cell所使用，且有expansion的現象。 我們相信目前所得到的結果，已帶領我們走向正確的了解致病的TCR，下一步將走向老鼠的實驗系統，製造出帶有此類TCR的transgenic mice，以確定此些TCR對導致肌無力所扮演的角色，並進而發展出對此類自體免疫疾病的專一療法。
並列摘要
Myasthenia gravis (MG) is an autoimmune disease mediated by autoantibody against acetylcholine receptor (AchR). These antibodies blocked the signal transduction between neuromuscular junctions and resulted in muscle weakness. In this study, we analyzed the Vδ5- and V?-containing T cell receptor (TCR) ?chain gene usages, and the nucleotide sequences of the CDR3 region in MG patients and healthy controls. The aim is to find potential pathogenic T cells to aid the development of specific therapy for the disease of myasthenia gravis. In the study, cDNA of peripheral blood lymphocytes (PBLs) were made from four MG patients & three healthy controls (HCs). Additionally, the thymus and thyroid surgically removed and obtained from one of the MG patients were also included in this study. TCR genes carrying Vδ5 or V? were specifically amplified from cDNA by C?specific primer paired with Vδ5- and V?- specific primers, respectively. The PCR products were then subcloned and sequenced to analyze the nucleotide sequences of the CDR3 region. First of all, in the Vδ5-C?gene usage, we observed a significant differential usage on TCRs with charged amino acids on the sixth or seventh position of the CDR3 region between patients and healthy controls. This result unexpectedly correlates well with the data obtained from experimental autoimmune myasthenia gravis (EAMG) and strongly indicates these TCRs are related to the development of the MG. Second of all, in the V?-C?gene usage, there were no significant sequence differences in the CDR3 region between MG patients and the HC, except MG-Ⅳ, which did not use invariant TCR bearing V? sequences in our data. In addition, we also found a new third invariant TCR ?chain in human—V?-J?2-C? This type of TCR was detected as an expanded population in CD4+CD8+ DP T cells from one of the MG patients. We believe that our data do shed light on understanding the TCR usage in MG patients and the potential pathogenic TCRs. Generating mice carrying our TCR ?chain sequences is the next step in order to confirm the role of the T cells bearing our TCRs. To help developing the specific therapy to medicate myasthenia gravis will be our final goal.
參考文獻
延伸閱讀
- 徐孟達（1998）。致活性T細胞和運動相關之探討。中華體育季刊，12(2)，86-93。https://doi.org/10.6223/qcpe.1202.199809.1712
- Liao, T. L. (2010). A型流感病毒核蛋白泛素化/去泛素化修飾與病毒核醣核酸複製調控機制之研究 [doctoral dissertation, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU.2010.02948
- Chuang, K. W. (2017). 肢帶型肌肉失養症所引起之心臟功能異常與NRIP(細胞核受體交互作用蛋白質)異常調控的關係 [master's thesis, National Taiwan University]. Airiti Library. https://doi.org/10.6342/NTU201703073
- Xue, W. (2004). Molecular cytogenetic, epigenetic and tissue dynamic study of gestational trophoblastic disease [doctoral dissertation, The University of Hong Kong]. Airiti Library. https://www.airitilibrary.com/Article/Detail?DocID=U0029-1812201200018133
- 吳哲維（2008）。Association of Gene Polymorphisms in DNA Base-Excision Repair Pathway with Differentiated Thyroid Cancer〔碩士論文，高雄醫學大學〕。華藝線上圖書館。https://doi.org/10.6832/KMU.2008.00065