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研究生:張瑞瑾
研究生(外文):Jui-Chin Chang
論文名稱:經活化Integrina6b1引發人類血小板形態改變之研究
論文名稱(外文):Morphological and Biochemical Analyses in Human Platelets Activated Through Integrin a6b1
指導教授:羅時成羅時成引用關係
指導教授(外文):Szecheng J. Lo
學位類別:博士
校院名稱:國立陽明大學
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2005
畢業學年度:94
語文別:英文
論文頁數:110
中文關鍵詞:血小板形態改變細胞骨骼肌動蛋白
外文關鍵詞:integrinplateletlaminin receptoractin-cytoskeletonfilopodiumCdc42PI3Krhodostominmorphological changesignal transduction
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血小板的主要生理功能在於凝血,而其中細胞膜上的吸附性蛋白受體integrin在此扮演著重要的角色。血小板可藉由integrin吸附到血管受傷的部位,之後又可由integrin 的作用,促使血小板凝集,而達成止血的目的。在血小板的研究中,經由活化integrin aIIbb3和a2b1,使得血小板活化並造成形態改變的機制,已有相當的了解。然而,integrin a6b1在血小板上的功能卻鮮少有報告。
為了瞭解血小板上integrin a6b1的作用,我們以抗integrin a6b1的單株抗體 GoH3和 laminin作為介質(substrate) ,進行一系列細胞形態和生化的分析。同時,我們也使用可活化integrin aIIbb3的重組蛇毒蛋白GST-rhodostomin作為對照,以瞭解這兩種integrin在血小板上的差異。在形態的分析中,藉由掃描式電子顯微鏡(SEM)和干涉相位差(DIC)顯微鏡可觀察到,血小板可在15分鐘內就吸附並完全伸展(fully spread)於以GST-rhodostomin為介質的玻片上;相對的,血小板雖然會黏貼在以GoH3為介質的玻片上,卻形成許多filopodia和lamellipodia的延伸,並不會完全伸展,即便是吸附時間超過一小時也是如此。在以螢光顯微鏡和穿透式電子顯微鏡(TEM)分析細胞骨骼(cytoskeleton)肌動蛋白(actin)在血小板中的分布時,可見當血小板黏貼伸展於GST-rhodostomin的介質上時,肌動蛋白成束分布於血小板邊緣及中央部位;而當血小板黏貼於GoH3的介質時, 肌動蛋白束由血小板中央向外呈現放射狀分布。然而,血小板的這些形態改變及肌動蛋白分布都會被Src 和PI3K的抑制劑所干擾,而有更多filopodia形成。經由免疫沉澱及西方墨點法等生化分析,在血小板吸附於GST-rhodostomin及GoH3這兩種介質時,Src及Syk等都會被活化。另外,當血小板吸附於GoH3的介質上,PI3K的活性和p110次單位之酪氨酸(tyrosine)被磷酸化(phosphorylation)的程度都較黏貼在GST-rhodostomin介質上高。由於細胞形態改變受到GTPase Rho家族的調控,因此當進一步分析GTPase的活性時發現,在血小板吸附於GoH3介質時,Cdc42的活化程度較黏貼於GST-rhodostomin介質高;而且PI3K的抑制劑wortmannin可以再提高Cdc42和Rac的活化程度。由此推論,當血小板上的integrin a6b1被活化時,會使Rac/Cdc42活化,造成血小板形態的改變;而Rac/Cdc42的活化程度又受PI3K的活性所調控。
Platelets play an essential role of hemastasis in prevention of bleeding from damaged blood vessels. Integrins, a superfamily of adhesion receptors, are involved in platelet activation and aggregation by interaction with exposed subendothelial matrix and secreted extracellular matrix, respectively. The platelet morphological changes and activations through integrin aIIbb3 and a2b1 are well characterized, but that via integrin a6b1 is less understood.
To examine the activation of platelets through integrin a6b1, anti-integrin a6 mAb, GoH3, were used as substrates. GST-rhodostomin, a recombinant snake disintegrin which activates integin aIIbb3, was parallel used as a comparison. Results of scanning electronic microscopy (SEM) and differential interference contrast (DIC) microscopy showed that platelets formed filopodia and lamellipodia but never fully spread on plates coated with laminin or GoH3; in contrast, platelets fully spread on GST-rhodostomin substrate within 15 minutes. By using fluorescence microscopy and whole-mount TEM to analyze the actin cytoskeleton distribution, we found that actin formed bundles in the peripheral and central portion of platelets when they adhered on GST-rhodostomin while actin were radiated out from the central region of platelets when they adhered on GoH3 mAb. Platelets treated with Src kinase inhibitor (PP1) and PI3 kinase inhibitors (Wortmannin and LY294002) showed that the morphology and actin patterns were disturbed both on GoH3 mAb- and GST-rhodostomin-coated plates. Results of immunoprecipitation and immunoblotting showed that Src and Syk were activated when platelets adhered on both of GST-rhodostomin and GoH3 mAb substrates. And a higher phosphotyrosine signal of PI3K catalytic subunit p110 and a higher PI3K activity were detected when platelets adhered on GoH3 mAb than they spread on GST-rhodostomin substrates. To further investigate the small GTPase Rho family participated in inducing different morphology of platelets through integrin aIIbb3 and a6b1 activation, small GTPase activity assay was performed. A higher amount of activated Cdc42 was detected when platelets adhered on GoH3 mAb than they spread on GST-rhodostomin substrate. The activity of Cdc42 and Rac was increased in platelet pretreated with PI3K inhibitor, wortmannin. Taken together, the results suggested that PI3K is a modulator to regulate the level of activated Rac/Cdc42 in filopodia formation of platelets which induced by a6b1 integrin.
Content

Abstract ------------------------------------------ 1
中文摘要 ------------------------------------------ 3
Introduction -------------------------------------- 5
Materials ----------------------------------------- 21
Methods ------------------------------------------- 32
Results ------------------------------------------- 41
Discussion ---------------------------------------- 51
References ---------------------------------------- 60
Figures ------------------------------------------- 79
Appendix ------------------------------------------ 109
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