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請用此 Handle URI 來引用此文件: http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33054
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dc.contributor.advisor莊寧寧
dc.contributor.authorJin-Wen Huangen
dc.contributor.author黃勁文zh_TW
dc.date.accessioned2021-06-13T04:23:20Z-
dc.date.available2006-07-25
dc.date.copyright2006-07-25
dc.date.issued2006
dc.date.submitted2006-07-23
dc.identifier.citationAngenstein, F., Evans, A. M., Settlage, R. E., Moran, S. T., Ling, S. C., Klintsova, A. Y., Shabanowitz, J., Hunt, D. F., & Greenough, W. T. (2002). A receptor for activated C kinase is part of messenger ribonucleoprotein complexes associated with polyA-mRNAs in neurons. J Neurosci, 22(20), 8827-8837.
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Clasper, S., Vekemans, S., Fiore, M., Plebanski, M., Wordsworth, P., David, G., & Jackson, D. G. (1999). Inducible expression of the cell surface heparan sulfate proteoglycan syndecan-2 (fibroglycan) on human activated macrophages can regulate fibroblast growth factor action. J Biol Chem, 274(34), 24113-24123.
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Geijsen, N., Spaargaren, M., Raaijmakers, J. A., Lammers, J. W., Koenderman, L., & Coffer, P. J. (1999). Association of RACK1 and PKCbeta with the common beta-chain of the IL-5/IL-3/GM-CSF receptor. Oncogene, 18(36), 5126-5130.
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Kiely, P. A.Leahy, M.O'Gorman, D.O'Connor, R.(2005). RACK1-mediated integration of adhesion and insulin-like growth factor I (IGF-I) signaling and cell migration are defective in cells expressing an IGF-I receptor mutated at tyrosines 1250 and 1251 J Biol Chem, 280(9), 7624-33
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dc.identifier.urihttp://tdr.lib.ntu.edu.tw/jspui/handle/123456789/33054-
dc.description.abstract在本實驗中,我們利用重組蛋白和親和管柱進行細胞外(in vitro)的實驗,發現重組syndecan-2和RACK1有交互作用,而在細胞內(in vivo)實驗,經免疫沉澱法和細胞免疫染色法也證實RACK1的確存在於syndecan-2蛋白複合體中,且兩分子的交互作用主要位於細胞膜上。進一步探討syndecan-2和RACK1的作用,我們發現當syndecan-2第180位置的酪胺酸在磷酸化狀態下,和RACK1的交互作用有增強的現象。
另一方面,我們以已知會造成syndecan-2酪氨酸磷酸化的細胞素(cytokine),巨噬細胞群叢形成促進因子(GM-CSF)處理細胞,syndecan-2和RACK1的作用不論是有無磷酸化的參與(phosphate-dependent or independent)皆有增強的現象,且有時間和劑量上的效應(time-and dose-dependent effect),該現象推測可能是與syndecan-2的磷酸化及syndecan-2的叢聚現象(clustering)所造成,但仍需更進一步實驗證實。
最後,我們試著分析syndecan-2在細胞生理的功能,並探討與RACK1交互作用的相關性,來進一步了解這兩個分子交互作用可能的細胞生理意義。由上述實驗結果,我們知道syndecan-2胞內區域磷酸化會增加兩分子間的作用,因此我們建構了正常型(wild type:Syn-2-wt)、酪胺酸單點突變(Syn-2-Y180F、Syn-2-Y192F、Syn-2-Y180/192F)及胞內區域(intracellular domain:PEP Syn-2-cyto) 的syndecan-2表現載體,在細胞中進行表現後,分別以細胞型態、貼附、爬移和增生等生理現象和生化方式進行觀察和分析,我們發現在不同載體表現的轉染細胞中,其細胞生理現象的差異和syndecan-2 /RACK1磷酸化參與交互作用的部分具有關聯性,然而是否藉由該作用的差異性來調控可能的分子(如:Src kinase or Protein kinase c)進而造成細胞生理現象的差異,其中的分子機轉仍須進一步的實驗證明。在本實驗中雖然無法明確了解syndecan-2和RACK1交互作用的意義,但仍提供了將來可能的實驗方向。
zh_TW
dc.description.abstractIn this study, RACK1 (Receptor for Activated C Kinase 1) was found to be reactive with syndecan-2 in vitro and in vivo. Through affinity column chromatography and immunoprecipitation analysis as well as immunocytochemical colocalization studies, the reaction between RACK1 and syndecan-2 was evidenced in BALB/3T3 cells. Recombinant syndecan-2 and PEP Syn-2-cyto were applied to demonstrate that tyrosine 180 of syndecan-2 is a targeted site for Src tyrosine kinase and the reaction with RACK1 is enhanced after this tyrosine phosphorylation. In parallel, when granulocyte-macrophage colony-stimulating factor (GM-CSF) was applied to activate cellular tyrosine kinase of HeLa cells, a significant positive interaction was revealed between syndecan-2 and RACK1 with time and dose-dependence.
HeLa cells were further subject to transfections with wild type and mutant syndecan-2 vectors(Syn-2-Y180F、Syn-2-Y192F、Syn-2-Y180/192F) to show that the reaction of syndecan-2 with RACK1 was suppressed when tyrosine 180 phosphorylation site was absent. To elucidate the physiological significance of this selective reaction between syndecan-2 and RACK1, studies with adhesion, migration, and poliferation were focused. HeLa cells transfected with Syn-2-Y180F mutant vectors exhibited less motile and adhesion ability. Furthermore, the morphology of Syn-2-Y180F transfants became round and refractile. These results imply that tyrosine 180 of syndecan-2 may involve in the cytoskeleton organization, focal contacts formation, and tyrosine kinase regulation through selective reaction with RACK1.
en
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dc.description.tableofcontents中文摘要..................................................1
ABSTRACT..................................................2
壹、前言
1.Syndecans 家族的特性...................................3
2.Syndecan-2的基本特性....................................4
3.RACK1的基本特性.........................................6
4.實驗動機................................................7
5.實驗架構................................................7
貳、器材與藥品
1. 生物材料...............................................8
2. 實驗材料...............................................8
3. 實驗設備...............................................8
4. 實驗藥品與試劑.........................................9
5. 引子的設計............................................14
參、實驗方法
抽取總量RNA..............................................15
置備cDNA.................................................15
聚合酶連鎖反應...........................................16
DNA 電泳和EtBr染色.......................................16
純化DNA產物..............................................17
syndecan-2 cDNA引子的設計與全長和胞內功能區域DNA片段的獲得.......................................................17
質體的構築...............................................18
構築小鼠全長與胞內功能區域syndecan-2 cDNA在細菌表現質體的
保存與定序...............................................19
小鼠全長與胞內功能區域syndecan-2 cDNA在細菌表現質體的構築19
構築全長與胞內功能區域syndecan-2 cDNA在哺乳動物細胞表現質
體的保存與序.............................................20
全長與胞內功能區域syndecan-2 cDNA在哺乳動物細胞表現質體的
構築.....................................................21
單鹼基點突變小鼠syndecan-2 cDNA 在細菌和哺乳動物細胞表現
質體的構築...............................................21
抽取小量質體.............................................23
細菌重組蛋白質的誘導表現.................................23
全長、胞內功能區域syndecan-2 和RACK1重組蛋白的分離與純化24
蛋白質定量...............................................25
Tricine-硫酸十二酯鈉聚丙烯醯氯膠電泳分析.................25
Coomassie blue R-250 染色法..............................26
西方點墨分析法...........................................26
細胞培養基...............................................26
細胞培養.................................................27
全長syndecan-2及其點突變株的細胞轉染.....................27
篩選轉染有全長syndecan-2及其點突變株的細胞株群...........28
細胞免疫染色.............................................28
細胞膜的粗萃.............................................28
免疫沉澱法...............................................29
細胞進行粒細胞/巨噬細胞群叢形成促進因子的處理............29
Hitrap-PEP taxol 和syndecan-2親和管柱的製備與操作........30
Hitrap- syndecan-2-PEP cyto 或RACK1 鎳離子嵌合親和管柱的
製備與作.................................................31
重組Src (p60c-src) 對syndecan-2、syndecan-2-Y180F
syndecan-2-PEP cyto的磷酸化作用..........................31
細胞增生分析.............................................32
細胞貼附分析.............................................32
細胞遷移能力分析.........................................33
肆、結果
全長和胞內syndecan-2 cDNA 的選殖以及點突變的建構和定序...34
小鼠全長和胞內syndecan-2重組蛋白的表現和純化.............34
以親和管柱色層分析證實重組蛋白RACK1和全長及胞內syndecan-2
具有交互作用的現象.......................................35
以親和管柱色層分析細胞中RACK1和syndecan-2-PEPCYTO重組蛋白
的交互作用,和免疫沉澱法證實syndecan-2和RACK1 complex 在
細胞中的存在.............................................36
以共軛焦顯微鏡觀察syndecan-2和RACK1在細胞中的重疊位置....36
syndecan-2-PEP cyto、syndecan-2和syndecan-2-Y180 F進行Src(p60 c-src)的磷酸化作用................................37
syndecan-2-PEP cyto在Src(p60 c-src)的磷酸化作用下有增強
與RACK1交互作用的現象....................................37
HeLa細胞在GM-CSF處理下,syndecan-2和RACK1交互作用有增強的
現象.....................................................38
HeLa細胞轉染syndecan-2基因作表現,觀察細胞型態...........39
HeLa細胞轉染syndecan-2及其突變株作基因表現後,分別進行細
胞貼附分析...............................................39
HeLa細胞轉染syndecan-2的突變株作基因表現後,觀察細胞型態
的差異...................................................40
HeLa細胞轉染syndecan-2及其突變株作基因表現後,分別進行細
胞遷移能力試驗...........................................41
HeLa細胞轉染syndecan-2及其突變株作基因表現後,進行細胞增
生分析...................................................42
HeLa細胞轉染syndecan-2胞內區域,觀察細胞型態的差異.......43
HeLa細胞轉染syndecan-2及其突變株作基因表現後,粗萃細胞膜
後分別進行免疫沉澱法.....................................43
伍、討論.................................................45
陸、參考文獻.............................................53
柒、圖表.................................................60
dc.language.isozh-TW
dc.subjectSyndecan-2zh_TW
dc.subjectRACK1zh_TW
dc.title探討syndecan-2和RACK1的交互作用與其細胞生理意義之研究zh_TW
dc.titleThe functional role of syndecan-2 in the molecular interaction with RACK1 in cellsen
dc.typeThesis
dc.date.schoolyear94-2
dc.description.degree碩士
dc.contributor.coadvisor齊肖琪
dc.contributor.oralexamcommittee潘建源,黃偉邦
dc.subject.keywordSyndecan-2,RACK1,zh_TW
dc.relation.page91
dc.rights.note有償授權
dc.date.accepted2006-07-23
dc.contributor.author-college生命科學院zh_TW
dc.contributor.author-dept動物學研究研究所zh_TW
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