賴明宗Lai, Ming-Zong臺灣大學:免疫學研究所黎于綺Lee, Yu-ChiYu-ChiLee2010-05-102018-07-092010-05-102018-07-092009U0001-1908200912012700http://ntur.lib.ntu.edu.tw//handle/246246/181816Paxillin 是大小為68-kD的細胞骨架轉接蛋白，可連結至focal adhesion複合體。它具有許多可和其他蛋白交互作用的區塊，並藉此整合外來訊息並加以傳遞。近年來有許多研究指出，paxillin 蛋白上絲胺酸及酪胺酸的磷酸化情形對其功能相當重要，但paxillin的磷酸化在T細胞的活化上所扮演的角色目前並不清楚。在本研究中，我們建立表現 paxillin 上FAK、JNK及p38 MAPK磷酸化位置突變蛋白的 DO11.10 細胞株，探討paxillin對 T細胞活化的影響。我們的實驗中發現，雖然paxillin 在上皮細胞及神經元細胞的延展及遷徙扮演關鍵角色，在 DO11.10 細胞中表現 paxillin 的突變株並不影響細胞的integrin黏附到其基質的能力，且只有表現FAK 磷酸化位置 paxillin 突變株細胞之遷徙能力受到些微的抑制。雖然JNK 和 p38 MAPK 磷酸化 paxillin 並不影響T細胞的移動，在表現 JNK/p38MAPK 雙磷酸化位置paxillin 突變株的 DO11.10 細胞中，發現TCR 活化後 IL-2 的產量明顯減少，而這個現象可歸因於轉錄因子NFAT的進核受到抑制。了更深入探討paxillin 對於正常T細胞的活化有何影響，我們也建立表現 paxillin磷酸化位置突變蛋白的基因轉殖小鼠。和同胎對照小鼠相比，基因轉殖小鼠的T細胞的發育並無明顯差異，細胞的黏附和遷徙也不受影響，但活化後細胞增生的速度及產生IL-2的能力都受到抑制。在基因轉殖小鼠中同樣可觀察到NFAT 進入細胞核的量明顯減少，除了影響IL-2 的分泌，IFN-gamma 及 IL-4 的產生也受到影響。合以上實驗結果，同時在paxillin的p38MAPK及JNK 磷酸化位置進行突變，可明顯抑制T細胞的活化。而被p38 MAPK 及 JNK所活化的paxillin 是如何調控NFAT 的進核以及T細胞的活化，則有待進一步的實驗探討。Paxillin is a 68-kD cytoskeletal adaptor protein associated with focal adhesion complex. It is a multidomain adaptor that facilitates signal integration and transduction. Recent studies revealed critical roles for tyrosine and serine phosphorylation of paxillin by FAK, JNK and p38 MAPK, but the consequence of paxillin phosphorylation in T cells remains unknown. In this study, we addressed this issue by overexpressing paxillin mutants with respective phosphorylation sites of FAK, JNK, and p38 MAPK in T cells, and examined the role of paxillin phosphorylation in T cell activation, adhesion, and migration.ontradictory to the reported effect on epithelial cells and neurite, all the paxillin mutants examined did not interfere with integrin-mediated T-cell adhesion. Overexpression of paxillin with mutations at phosphorylation sites of FAK (Y31F/Y118F) in T cells reduced SDF-1alpha--stimulated migration, but did not affect T cell activation, and other parameters of T cell activation remain normal. In contrast, overexpression of paxillin with double mutations at phosphorylation sites of p38 MAPK and JNK (S85A/S178A) in T cells did not alter cell adhesion and migration, but effectively suppressed IL-2 production, the signature of T cell activation. Inhibition by [S85A/S178A]-paxillin could be partly attributed to a selective suppression of NFAT activation.o study the role of paxillin in T cell activation in normal T cells, we further generated [S85A/S178A]-paxillin transgenic mice. Although no significant effect of [S85A/S178A]-paxillin on T cell development was observed, both T cell proliferation and IL-2 production were suppressed in transgenic mice compared to NLC mice. In addition, NFAT translocation was partially interfered by [S85A/S178A]-paxillin transgene. n summary, phosphorylations of paxillin by different kinases play different roles in T cells and non-T cells. How [S85A/S178A]-paxillin modulates T cell activation and NFAT activation will be further investigated.摘要................................................................................................................................ii 錄...............................................................................................................................vi 一章 緒論...............................................................................................................1 .1 Paxillin..................................................................................................................1 .2 Paxillin Superfamily..............................................................................................2 .3 Paxillin的結構......................................................................................................3 .4 Paxillin的磷酸化..................................................................................................4 .5 Paxillin與 integrin................................................................................................5 .6 T細胞的活化........................................................................................................6 .7 Paxillin對於T細胞活化的初步研究結果.............................................................7 .8 研究方向與目的.....................................................................................................7二章 材料與方法..................................................................................................8.1 細胞株與細胞培養...............................................................................................8.1.1 細胞株................................................................................................................8.1.2 小鼠胸腺、脾臟細胞..........................................................................................8..1.3 細胞培養...........................................................................................................8.2 藥品與試劑...........................................................................................................8.3 抗體.......................................................................................................................9.4 質體構築...............................................................................................................9.5質體DNA 的轉染(transfection) ...........................................................................10.5.1 Calcium phosphate 轉染法……………………………………………………10.5.2反轉錄病毒感染法 (retroviral infection) ..........................................................10.6西方點墨法 (Western blot) ...................................................................................11.7 IL-2 產量分析.......................................................................................................11.8 Wound-healing assay..............................................................................................12.9 Conjugation assay...................................................................................................12.9.1 細胞染色............................................................................................................12.9.2 細胞結合............................................................................................................13.10 全細胞萃取液 (total cell lysate) 的製備..........................................................13.11 核萃取液 (nuclear extract) 的製備...................................................................13.12 Adhesion assay.....................................................................................................14.13 Chemotaxis assay.................................................................................................14.14 建立CD2-paxillin S178A/S85A 基因轉殖鼠.....................................................15.14.1Genomic DNA 測試..........................................................................................15.14.1.1純化小鼠之Genomic DNA............................................................................15.14.1.2 篩選待有轉殖基因之小鼠...........................................................................15.14.2.1 RNA的純化...................................................................................................16.14.2.2反轉錄與聚合酶連鎖反應( RT- PCR) .........................................................16.15 CD2-paxillin S178A/S85A 基因轉殖鼠之分析................................................17.15.2.1 T 細胞增殖分析...........................................................................................17.15.2.2 IL-2產量分析...............................................................................................18.15.2.3細胞表面染色分析........................................................................................18三章 研究結果......................................................................................................19 .1 表現 paxillin 磷酸化位置突變株對T細胞活化時產生IL-2能力之影響…...19.2 Paxillin的JNK/p38MAPK磷酸化位置突變會影響轉錄因子NFAT的進核..20.3 表現 paxillin 磷酸化位置突變株不影響細胞的黏附(adhesion)……………..21.4 表現paxillin FAK磷酸化位置突變株會抑制細胞的遷移…………………….22.5 表現Paxillin磷酸化位置突變株不影響 T細胞與 B細胞的結合…………..22.6 Paxillin 磷酸化位置突變株會影響NIH-3T3移動的能力……………………..23.7 建立paxillin 的JNK/p38MAPK雙磷酸化位置突變的基因轉殖小鼠………24.8 Paxillin 的JNK和p38MAPK雙磷酸化位置突變的基因轉殖小鼠T細胞數量與發育之分析………………………………………………………………………..24.9 表現 paxillin S178A/S85A雙磷酸化位置突變蛋白的基因轉殖小鼠的胸腺細胞和脾臟細胞經活化後，細胞增生及 IL-2的分泌量減少………………………..25.10 表現 paxillin S178A/S85A雙磷酸化位置突變蛋白的基因轉殖小鼠之T細胞經活化後黏附及遷移的能力皆不受影響…………………………………………..26.11 表現 paxillin S178A/S85A雙磷酸化位置突變蛋白的基因轉殖小鼠之T細胞經活化後，細胞核內NFAT轉錄因子的量減少……………………………………26.12 表現 paxillin S178A/S85A雙磷酸化位置突變蛋白的基因轉殖小鼠之T細胞經活化後，產生之IFN-及IL-4的量減少…………………………………………..27四章 結果討論......................................................................................................29圖表.............................................................................................................................32 考文獻......................................................................................................................60application/pdf2039159 bytesapplication/pdfen-UST細胞paxillin磷酸化T cellphosphorylationhttp://ntur.lib.ntu.edu.tw/bitstream/246246/181816/1/ntu-98-R96449013-1.pdf