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???org.dspace.app.webui.jsptag.ItemTag.dcfield??? | Value | Language |
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dc.contributor.advisor | 賴明宗(Ming-Zong Lai) | |
dc.contributor.author | An-Yun Chang | en |
dc.contributor.author | 章安雲 | zh_TW |
dc.date.accessioned | 2021-06-13T06:58:23Z | - |
dc.date.available | 2005-08-04 | |
dc.date.copyright | 2005-08-04 | |
dc.date.issued | 2005 | |
dc.date.submitted | 2005-07-27 | |
dc.identifier.citation | 參考文獻
Brown, M. C. and C. E. Turner (2004). 'Paxillin: adapting to change.' Physiol Rev 84(4): 1315-39. Doucey, M. A. and D. F. Legler et al. (2003). 'The β1 and β3 integrins promote T cell receptor-mediated cytotoxic T lymphocyte activation.' J Biol Chem 278(29): 26983-91. Garcia, G. G. and R. A. Miller (2002). 'Age-dependent defects in TCR-triggered cytoskeletal rearrangement in CD4+ T cells.' J Immunol 169(9): 5021-7. Glenney, J. R., Jr. and L. Zokas (1989). 'Novel tyrosine kinase substrates from Rous sarcoma virus-transformed cells are present in the membrane skeleton.' J Cell Biol 108(6): 2401-8. Haaland, R. E., W. Yu, et al. (2005). 'Identification of LKLF-regulated genes in quiescent CD4+ T lymphocytes.' Mol Immunol 42(5): 627-41. Hagel, M., E. L. George, et al. (2002). 'The adaptor protein paxillin is essential for normal development in the mouse and is a critical transducer of fibronectin signaling.' Mol Cell Biol 22(3): 901-15. Hogg, N., M. L. Laschinger, et al. (2003). 'T-cell integrins: more than just sticking points.' J Cell Science 116: 4695-705. Huang, C., C. H. Borchers, et al. (2004). 'Phosphorylation of paxillin by p38MAPK is involved in the neurite extension of PC-12 cells.' J Cell Biol 164(4): 593-602. Huang, C., Z. Rajfur, et al. (2003). 'JNK phosphorylates paxillin and regulates cell migration.' Nature 424(6945): 219-23. Huppa, J. B. and M. M. Davis (2003). 'T-cell-antigen recognition and the immunological synapse.' Nature Rev. Immunol 3: 973-83. Ishibe, S., D. Joly, et al. (2003). 'Phosphorylation-dependent paxillin-ERK association mediates hepatocyte growth factor-stimulated epithelial morphogenesis.' Mol Cell 12(5): 1275-85. Ku, H. and K. E. Meier (2000). 'Phosphorylation of paxillin via the ERK mitogen-activated protein kinase cascade in EL4 thymoma cells.' J Biol Chem 275(15): 11333-40. Liu, S., M. Slepak, et al. (2001). 'Binding of Paxillin to the alpha 9 Integrin Cytoplasmic Domain Inhibits Cell Spreading.' J Biol Chem 276(40): 37086-92. Liu, S., S. M. Thomas, et al. (1999). 'Binding of paxillin to alpha4 integrins modifies integrin-dependent biological responses.' Nature 402(6762): 676-81. Liu, Z. X., C. F. Yu, et al. (2002). 'Hepatocyte growth factor induces ERK-dependent paxillin phosphorylation and regulates paxillin-focal adhesion kinase association.' J Biol Chem 277(12): 10452-8. Ostergaard, H. L., O. Lou, et al. (1998). 'Paxillin phosphorylation and association with Lck and Pyk2 in anti-CD3- or anti-CD45-stimulated T cells.' J Biol Chem 273(10): 5692-6. Romanova, L. Y., S. Hashimoto, et al. (2004). 'Phosphorylation of paxillin tyrosines 31 and 118 controls polarization and motility of lymphoid cells and is PMA-sensitive.' J Cell Sci 117(Pt 17): 3759-68. Rose, D. M., S. Liu, et al. (2003). 'Paxillin binding to the alpha 4 integrin subunit stimulates LFA-1 (integrin alpha L beta 2)-dependent T cell migration by augmenting the activation of focal adhesion kinase/proline-rich tyrosine kinase-2.' J Immunol 170(12): 5912-8. Sechi, A. S. and J. Wehland (2004). 'Interplay between TCR signalling and actin cytoskeleton dynamics.' TRENDS in Immunol 25: 257-65. Tang, D. D., C. E. Turner, et al. (2003). 'Expression of non-phosphorylatable paxillin mutants in canine tracheal smooth muscle inhibits tension development.' J Physiol 553(Pt 1): 21-35. Turner, C. E. (1998). 'Paxillin.' Int J Biochem Cell Biol 30(9): 955-9. Turner, C. E. (2000). 'Paxillin and focal adhesion signalling.' Nat Cell Biol 2(12): E231-6. Turner, C. E., J. R. Glenney, Jr., et al. (1990). 'Paxillin: a new vinculin-binding protein present in focal adhesions.' J Cell Biol 111(3): 1059-68. Vadlamudi, R., L. Adam, et al. (1999). 'Serine phosphorylation of paxillin by heregulin-beta1: role of p38 mitogen activated protein kinase.' Oncogene 18(51): 7253-64. Wang, Y. and T. D. Gilmore (2003). 'Zyxin and paxillin proteins: focal adhesion plaque LIM domain proteins go nuclear.' Biochim Biophys Acta 1593(2-3): 115-20. Zuckerman, L. A. and L. Pullen et al. (1998). 'Functional consequences of costimulation by ICAM-1 on IL-2 gene expression and T cell activation.' J Immunol 160: 3259-68. | |
dc.identifier.uri | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/35558 | - |
dc.description.abstract | Paxillin是integrin下游重要的轉接蛋白(adaptor protein)之一。Integrin 會幫助T細胞和抗原呈獻細胞的結合,且在T細胞活化時提供共同活化訊息(co-stimulatory signal)。已知paxillin會在T細胞活化過程中被磷酸化,且聚集在immunological synapse的位置。然而paxillin如何參與T細胞的活化,並沒有明確的結論。在本論文中,我們建立表現paxillin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。也建立表現paxillin磷酸化位置突變蛋白的基因轉殖小鼠,於正常T細胞中分析paxillin在T細胞發育及活化所扮演的角色。
研究中發現,表現paxillin的FAK、JNK或p38MAPK單一磷酸化位置突變蛋白的DO11.10 T細胞株,活化後IL-2的產生或與B細胞結合的能力,皆和YFP對照細胞相當。在表現不同組合的paxillin雙磷酸化位置突變蛋白的DO11.10 T細胞株,TCR活化後分泌IL-2的量減少。其中尤其以表現JNK/p38MAPK雙磷酸化位置突變(S178A/S85A)的paxillin影響IL-2產生最顯著。但paxillin 的雙磷酸化位置突變不影響T細胞與B細胞結合能力。 爲探討paxillin在正常T細胞發育及活化所扮演的角色,我們更進一步建立表現paxillin雙磷酸化位置突變(S178A/S85A)蛋白的基因轉殖小鼠。與同胎對照小鼠相比,發現基因轉殖小鼠的T細胞發育有缺陷,胸腺single positive T細胞及脾臟CD4 T細胞族群均降低。另外,S178A/S85A-paxillin基因轉殖小鼠的胸腺或脾臟T細胞,TCR刺激後細胞增生及IL-2分泌,都比同胎對照小鼠減少。 綜合我們的結果,paxillin在p38MAPK、JNK或FAK磷酸化位置的突變組合,明顯抑制T細胞株的活化。阻斷paxillin被p38MAPK及JNK同時磷酸化也抑制正常T細胞的發育和活化。我們的結果顯示paxillin被不同激酶磷酸化,尤其是p38MAPK及JNK,是T細胞活化及發育所必需。 | zh_TW |
dc.description.abstract | Paxillin is one of the important adaptor proteins down stream of integrin. Integrin supports the association of T cell and antigen presenting cell, and provides co-stimulatory signal during T cell activation. It is known that during T cell activation, paxillin is phosphorylated and recruited to the site of immunological synapse. However, the exact role of paxillin in T cell activation has not been defined. In this study, we elucidated the role of paxillin in T cell activation, by expressing different paxillin phosphorylation site mutant in DO11.10 hybridoma. We also examined the role of paxillin in normal T cell activation and development, by expressing S178A/S85A paxillin double phosphorylation site mutant in transgenic mice.
Results from our study demonstrate that overexpression of paxillin with mutation at single phosphorylation site targeted by FAK, JNK, or p38 in DO11.10 hybridoma did not affect T cell activation, as measured by IL-2 production or T cell-antigen presenting cell conjugation. Expression of paxillin with mutation at double phosphorylation site in different combination, resulted in decreased IL-2 production upon TCR stimulation. Expression of paxillin with mutation at double phosphorylation site targeted by JNK and p38 (S178A/S85A), led to the most significant reduction of IL-2 generation. However, T cell-antigen presenting cell conjugation was not altered by expression of paxillin with mutation at double phosphorylation sites. To study the role of paxillin in T cell activation and development in normal T cells, we further generated paxillin S178A/S85A transgenic mice. In comparison with normal littermate control mice, transgenic mice showed defects in T cell development, with reduction in thymic single positive T cell population and in splenic CD4 T cell population. Thymocytes and splenic T cells from S178A/S85A-paxillin transgenic mice, displayed attenuated cell proliferation as well as IL-2 production during TCR stimulation. In summary, mutation of paxillin at phosphorylation sites targeted by combination of p38MAPK, JNK, or FAK clearly inhibited activation of T cell line. In addition, blockage of paxillin phosphorylation by p38MAPK and JNK suppressed activation and development of normal T cells. Our results indicate that phosphorylation of paxillin by different kinases, especially p38MAPK and JNK, is essential for T cell activation and T cell development. | en |
dc.description.provenance | Made available in DSpace on 2021-06-13T06:58:23Z (GMT). No. of bitstreams: 1 ntu-94-R92449002-1.pdf: 1112516 bytes, checksum: d99935c21d1acd512ef9e0f80a5fc15d (MD5) Previous issue date: 2005 | en |
dc.description.tableofcontents | 摘要…………………………………………………………….…...........ii
目錄……………………………………………………………...…........vi 第一章 緒論……………………………………………………….……1 1.1 Paxillin之簡介………………………………………………….......1 1.2 Paxillin的結構………………………………………………….…..2 1.3 Paxillin的功能..……………………………………………….……2 1.4 Paxillin和T細胞的關係……………………………………...……4 1.5 Paxillin和MAP Kinase的關係…………...…………………..……5 1.6 T細胞的活化………………………………………………….……6 1.7 Integrin和T細胞的關係……………………………………..….…7 第二章 材料及方法……………………………………………….........9 2.1 細胞株與細胞培養……………………………………………...….9 2.1.1 細胞株………………………………………………………..9 2.1.2 小鼠胸腺、脾臟與淋巴結細胞…………………………...….9 2.1.3 細胞培養……………………………………………….…….9 2.2 藥品與試劑………………………………………………………..10 2.3 抗體………………………………………………………………..10 2.4 質體構築…………………………………………………………..11 2.4.1 pcDNA4-paxillin WT-Myc、pcDNA4-paxillin Y118F-Myc pcDNA4-paxillin S178A-Myc、pcDNA4-paxillin S85A-Myc質體………………………………………………………....11 2.4.2 pcDNA4-paxillin Y118F/S178A-Myc、pcDNA4-paxillin Y118F/S85A-Myc、pcDNA4-paxillin S178A/S85A-Myc質體………………………………………………………..…12 2.4.3 pGC-paxillin WT-Myc-YFP、pGC-paxillin Y118F-Myc-YFP、pGC-paxillin S178A-Myc-YFP、pGC-paxillin S85A-Myc-YFP、pGC-paxillin Y118F/ S178A-Myc-YFP、pGC-paxillin Y118F/ S85A-Myc-YFP、pGC-paxillin S178A/ S85A-Myc-YFP 質體……………..12 2.4.4 CD2- paxillin S178A/S85A-Myc 質體………………...…13 2.5 質體DNA的轉染 (Transfection)………………………...………13 2.5.1 Calcium phosphate 轉染法……………………………..….13 2.5.2 反轉錄病毒感染法 (Retroviral infection)…………………13 2.6 西方點墨法 (Western Blot)………………………………………14 2.7 IL-2 產量分析 (IL-2 production assay)………………………….15 2.7.1 以抗CD3抗體活化DO11.10 paxillin突變株………….….15 2.7.2 以A20細胞呈獻OVA(323-339)胜肽活化DO11.10 paxillin突變株………………………………………………………16 2.8 T細胞與B細胞結合分析 ( Conjugation assay)…………………16 2.8.1 細胞染色……………………………………………………16 2.8.2 細胞結合……………………………………………………17 2.9 CD2-paxillin S178A/S85A 基因轉殖鼠之建立…………………17 2.9.1 Genomic DNA 測試……………………………………….17 2.9.2 RNA的純化……………………………………..………….18 2.9.3 反轉錄與聚合酶連鎖反應( RT- PCR)…………………….19 2.10 CD2-paxillin S178A/S85A 基因轉殖鼠之分析…..……………19 2.10.1 T細胞增殖分析……………………………………….…..19 2.10.2 IL-2產量分析…………..…………………………………20 2.10.3 細胞表面染色分析…………………………………….….20 第三章 研究結果…………………………………………………..….21 3.1 Paxillin單一磷酸化位置突變不影響T細胞活化中IL-2的產生.21 3.2 Paxillin雙磷酸化位置突變抑制T細胞活化中IL-2的產生…….22 3.3 Paxillin磷酸化位置突變不影響T細胞與B細胞的結合……….23 3.4 建立paxillin 的JNK/p38MAPK雙磷酸化位置突變的基因轉殖小鼠……………………………………………………………….….25 3.5 Paxillin 的JNK和p38MAPK雙磷酸化位置突變的基因轉殖小鼠分析……………..…………………………………………………26 3.5.1 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…………………………………………26 3.5.2 表現paxillin S178A/S85A雙磷酸化位置突變蛋白的基因轉殖小鼠胸線細胞染色分析……………...………………28 3.5.3 表現paxillin S178A/S85A雙磷酸化位置突變蛋白的基因轉殖小鼠脾臟細胞染色分析……………………………...29 第四章 結果討論………………………………………………..…… 31 圖表……………………………………………………………………...37 參考文獻…………………………………………………………...……62 | |
dc.language.iso | zh-TW | |
dc.title | Paxillin在T細胞活化上角色之探討 | zh_TW |
dc.title | The Role of the Adaptor Protein Paxillin in T Cell Activation | en |
dc.type | Thesis | |
dc.date.schoolyear | 93-2 | |
dc.description.degree | 碩士 | |
dc.contributor.oralexamcommittee | 李建國,王萬波 | |
dc.subject.keyword | T細胞,活化, | zh_TW |
dc.subject.keyword | Paxillin,T cell,activation, | en |
dc.relation.page | 66 | |
dc.rights.note | 有償授權 | |
dc.date.accepted | 2005-07-28 | |
dc.contributor.author-college | 醫學院 | zh_TW |
dc.contributor.author-dept | 免疫學研究所 | zh_TW |
Appears in Collections: | 免疫學研究所 |
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