果伽蘭臺灣大學：生化科學研究所王穗華WANG, SHUI-HUASHUI-HUAWANG2007-11-262018-07-062007-11-262018-07-062004http://ntur.lib.ntu.edu.tw//handle/246246/52749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。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.目錄……………………………………………………………………Ⅰ 中文摘要………………………………………………………………Ⅳ 英文摘要………………………………………………………………Ⅴ 縮寫表…………………………………………………………………Ⅵ 一、前言…………………………………………………………………1 1.1免疫系統……………………………………………………………1 1.2 T細胞和B細胞………………………………………………………1 1.2.1 T細胞……………………………………………………………1 1.2.2 B細胞……………………………………………………………3 1.3 T細胞抗原接受器(T Cell Recepter, TCR)與抗原(Antigen)以及主要組織相容性複合體(Major Histocompatibility Complex, MHC)………………………………………………………………………4 1.3.1 T細胞抗原接受器………………………………………………4 1.3.2 不變的T細胞接受器（Invariant TCR）………………………5 1.3.3 T細胞抗原接受器的基因座 (TCR loci)………………………6 1.3.4 T細胞接受器的V（D）J基因重組………………………………6 1.3.5 T細胞接受器與抗原以及主要組織相容性複合體(MHC)之間的作用………………………………………………………………………8 1.4 T細胞與自體免疫疾病……………………………………………8 1.5 肌無力症(Myasthenia Gravis)…………………………………10 1.5.1 神經末稍傳導途徑—乙醯膽鹼及其受器……………………10 1.5.2 EAMG（Experimental Autoimmune Myasthenia Gravis）…11 1.5.3 胸腺與肌無力症………………………………………………11 1.5.4 重症肌無力與T細胞抗原接受器………………………………12 1.5.5 目前治療肌無力症的主要方法………………………………13 1.6本論文所探討的主要問題…………………………………………13 二、實驗材料與方法…………………………………………………15 2.1實驗材料……………………………………………………………15 2.2實驗方法……………………………………………………………15 2.2.1分離出周邊淋巴球………………………………………………15 2.2.2引子(primer)的合成……………………………………………15 2.2.3 訊息核醣核酸（mRNA）與單股互補去氧核醣核酸（first strand cDNA）的製備…………………………………………………16 2.2.4訊息核醣核酸（mRNA）與單股互補去氧核醣核酸（first strand cDNA）的製備…………………………………………………16 2.2.5低熔點洋菜膠電泳分析（Low-melting Point Agarose Gel Electrophoresis）……………………………………………………17 2.2.6選植(Cloning)…………………………………………………17 2.2.6.1去氧核醣核酸的接合反應(DNA ligation)…………………17 2.2.6.2勝任細胞 (competent cell) 的製備………………………17 2.2.6.3轉型作用（Transformation）………………………………18 2.2.6.4 質體DNA的抽取………………………………………………………………………18 2.2.6.5核酸限制酶切割反應 (Digestion of Restriction Enzyme)…………………………………………………………………………19 2.2.6.6菌落聚合酶鍊鎖反應（colony-PCR）………………………19 2.2.6.7瓊酯明膠膠體電泳 (Agarose Gel Electrophoresis)……20 2.2.7核酸序列分析(Sequencing)……………………………………20 2.2.7.1循環反應（Cycle Reaction）………………………………20 2.2.7.2聚丙烯胺凝膠電泳分析（Polyacrylamide Gel Electrophoresis）……………………………………………………20 2.2.7.3自動核酸序列分析（Autosequencing）……………………21 三、結果………………………………………………………………22 3.1 V基因的使用情形…………………………………………………22 3.2 J基因的使用情形…………………………………………………23 3.2.1 Vδ5基因使用Jㄟ穧]的情形……………………………………23 3.2.2 V?基因使用Jㄟ穧]的情形……………………………………25 3.3 CDR3的性質………………………………………………………26 四、討論………………………………………………………………28 4.1 V基因的使用情形…………………………………………………28 4.2 J基因的使用情形…………………………………………………29 4.3 Clonal expansion………………………………………………30 4.4 CDR3……………………………………………………………………31 4.5 未來展望…………………………………………………………32 五、參考文獻…………………………………………………………34 圖表 表1. Table of PCR products studied of MG patients and healthy controls in Vδ5-C?and V?-C?genes………………38 表2.TCRVδ5-JΑNA and amino acid sequences 其CDR3 序列整理………………………………………………………………………39 表3.本實驗室所發現的其它條可能的Invariant TCR………………40 表4.在Vδ5基因的使用上，肌無力病人和正常人CDR3 junction區域帶有正電荷氨基酸的比率……………………………………………41 表5.CDR3序列分析整理—與線上基因庫比對的結果………………42 圖1.比較Vδ5和V?基因其Jㄟ穧]的使用情形………………………43 圖2.病人(MG)和正常人(HC)其Vδ5基因的使用下，其所銜接的Jㄟ穧]的使用情形……………………………………………………………44 圖3. 病人(MG)和正常人(HC)其V?基因的使用下，其所銜接的Jㄟ穧]的使用情……………………………………………………………45 圖4. J?usage of Vδ5 gene of HC-W/R/A & MG-D/Y/L…………46 圖5. J?usage of V? gene of HC-W/A & MG-D/Y/Ⅳ……………47 附錄一 T細胞接受器的V（D）J基因重組…………………………48 附錄二 T細胞與自體免疫疾病之間的關係…………………………49 附錄三 正常人與肌無力病人其神經傳導途徑—乙醯膽鹼受器之異同………………………………………………………………………50 附錄四 人類Jㄟ穧]的氨基酸序列…………………………………51 附錄五 EAMG（experimental autoimmune myasthenal gravis）體內所得的TCR ?錀DR3區域氨基酸分子的使用情形…………………53 附錄六 正常人(HC)和肌無力病人(MG)使用了Vδ5-C?& V?-Cㄓ劫細胞接受器CDR3區域的核酸與氨基酸序列 六之1.TCRVδ5-J DNA and amino acid sequences of clones from HC-2W mixed/CD4+/CD8+/ CD4- CD8- DN populations……………54 六之2 TCRVδ5-J DNA and amino acid sequences of clones from HC-1R mixed population ……………………………………………58 六之3 TCRVδ5-J DNA and amino acid sequences of clones from HC-2A mixed population……………………………………………59 六之4 TCRVδ5-J DNA and amino acid sequences of clones from MG-4D mixed population……………………………………………60 六之5 TCRVδ5-J DNA and amino acid sequences of clones from MG-6D mixed population……………………………………………61 六之6 TCRVδ5-J DNA and amino acid sequences of clones from MG-7D mixed population……………………………………………62 六之7. TCRVδ5-J DNA and amino acid sequences of clones from MG-2Y mixed /CD4+/CD8+/CD4+ CD8+ DP populations……63 六之8. TCRVδ5-J DNA and amino acid sequences of clones from MG-1L mixed/thymus/thyroid T cells………………………67 六之9. TCRV?-J DNA and amino acid sequences of clones from HC-2W mixed population………………………………………70 六之10. TCRV?-J DNA and amino acid sequences of clones from HC-2A CD4+/CD8+/CD4-CD8- DN populations………………71 六之11. TCRV?-J DNA and amino acid sequences of clones from MG-4D CD4+/CD8+/CD4-CD8- DN/CD4+ CD8+ DP populations……………………………………………………………74 六之12. TCRV?-J DNA and amino acid sequences of clones from MG-5D mixed population………………………………………77 六之13.TCRV?-J DNA and amino acid sequences of clones from MG-6D mixed population………………………………………78 六之14. TCRV?-J DNA and amino acid sequences of clones from MG-2Y CD4+/CD8+/CD4-CD8-DN/ CD4+ CD8+ DP populations79 六之15. TCRV?-JDNA and amino acid sequences of clones from MG-1Ⅳ mixed population……………………………………831701276 bytesapplication/pdfen-US自體免疫疾病肌無力T細胞接受器Autoimmune diseaseMyasthenia GravisT cell receptor[SDGs]SDG3otherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/52749/1/ntu-93-R91242011-1.pdf