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研究生:曾維廉
研究生(外文):Wei Lian Tseng
論文名稱:Reln蛋白於血小板中藉由改變PKCdelta及smallGTPase活性調控血小板功能
論文名稱(外文):A novel platelet protein Reln regulates platelet function through modulation of PKCdelta and small GTPase activity
指導教授:曾慶平
指導教授(外文):C. P. Tseng
學位類別:碩士
校院名稱:長庚大學
系所名稱:醫學生物技術研究所
學門:醫藥衛生學門
學類:醫學技術及檢驗學類
論文種類:學術論文
論文出版年:2008
畢業學年度:96
論文頁數:51
中文關鍵詞:血小板
外文關鍵詞:Reelinplatelet
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Reelin(Reln)是一個全長約420 kDa的extracellular protein。在過去的研究中已瞭解到Reln蛋白會調控神經的migration,對胚胎發育過程腦部的basal cortical的分層,以及成體神經的synapse plasticity中扮演關鍵的角色。除了神經系統外,在體內的circulation system中同樣也有Reln的存在。Reln不正常表現所導致的精神疾病如schizophrenia及bipolar disorder,也發現血液的Reln表現量也會產生改變,同時會有血小板活性異常的產生。然而Reln在血液系統中的功能卻仍不明瞭。在本研究顯示Reln在週邊血球細胞中會存在於血小板中的-granule,並且會隨血小板活化而釋放。而以外加Reln的方式也可發現Reln與血小板會有結合的情形。在血小板功能上,以外加Reln的方式可抑制血小板內PKCthr505磷酸化產生,並且活化Rac1。在以刺激劑活化血小板的過程中,Reln蛋白的存在同樣可以抑制PKC thr505活化,進一步分析血小板貼附到細胞外蛋白fibrinogen的過程中,外加Reln的情形下可以增加血小板貼附的面積,此過程可能藉由改變細胞內F-actin組成所致。在此我們了解到血液系統,特別於血小板上,Reelin蛋白確實存在其中,並且在血小板的影響上,Reln可能扮演著幫助血小板貼附的過程。
Reelin (Reln) is an extracellular matrix protein that plays a pivotal role in neuronal cell migration and is abundantly expressed in the plasma. However, the expression and function of Reln in the hematopoietic cells are barely characterized. In this study, we found that Reln was expressed in the megakaryocyte-like leukemic cell lines and human platelets. Subcellular localization analysis revealed that Reln distributed in the platelet a-granules and was released into the supernatant upon platelet activation. The lack of Reln mRNA in the platelet indicates that Reln was not generated endogenously by platelet. Instead, plasma could be the source for platelet. Consistent with these observations, we found that extracellular Reln recombinant protein is associated with the platelet. Platelet-Reln interaction was also revealed by a platelet adhesion assay toward the Reln-coated slides. During platelet adhesion to fibrinogen, the Reln-platelet interactions result in an increase in platelet spreading, lamellipodia formation and F-actin bundling. Such effects were diminished by the presence of Reln blocking antibody CR-50, indicating that Reln is a regulator of platelet spreading. Further analysis revealed that Reln signaling inhibited PKC activation and increased Rac1 ativity. Consistent with this observation, the Rac1 inhibitor NSC23766 reversed Reln effect on platelet spreading. These findings demonstrate for the first time that Reln plays a novel role in the control of platelet-fibrinogen interactions with modulation of PKC and Rac1 activity as the underlying mechanism.
目 錄
指導教授推薦書…………………………………………………………………
口試委員會審定書………………………………………………………………
授權書…………………………………………………………………………….......iii
誌謝……………………………………………………………………………….......iv
中文摘要……………………………………………………………………………....v
英文摘要………………………………………………………………………….......vi
目錄……………………………………………………………………………..........vii
第一章 緒論…………………………………………………………………….. 1
1.1 血小板………………………………………………….................................1
1.2 Reelin…………………………………………………………………..........2
1.3 Reelin在中樞神基系統的功能…………………………………………..…3
1.4 Reelin的致病機轉…………………………………………………………..5
1.5 Reelin與血液系統…………………………………………………………..6
第二章 材料與方法…………………………………………………..4
2. 1材料……………………………………………………………8
2.2 方法……………………………………………………………………8
第三章 結果………………………………………………………………………14
3.1分析Reln在血液系統中的表現情形………………………………………14
3.2 Reln在血小板內生合成方式………………………………………………15
3.3 Reln對於血小板功能的影響………………………………………………18
3.4以Reln重組蛋白處理血小板,分析在血小板內蛋白訊號的變化……..19
第四章 討論………………………………….....……………………………..21
第五章 參考文獻……………………………………………………….…….25
第六章 圖表……………………………………………………………………28

圖表目錄

Figure. 1建立研究Reln於血球細胞功能之cell line……………………………….28
Figure. 2比較不同血球細胞中Reln蛋白的表現情形 ……………………………29
Figure. 3利用immunoflourescence以confocal microscope觀察Reln在血小板中的
分佈位置…………………………………………………………………30
Figure. 4以sucrose gradient分離不同血小板fraction,觀察Reln在血小板中的分
佈位置……………………………………………………………………31
Figure. 5以agonist刺激血小板的活化,偵測Reln的釋放情形……………………32
Figure. 6利用分析血小板中mRNA及megakaryocyte中蛋白表現,探討血小板內
Reln的來源………………………………………………………………33
Figure. 7利用293T細胞大量表現Reln蛋白並純化……………………………… 34
Figure. 8外加Reln重組蛋白,觀察血小板與Reln間的交互作用………………35
Figure. 9加入Reln重組蛋白對於血小板凝集的影響…………………………… 36
Figure. 10 Reln重組蛋白對於血小板貼附過程的影響……………………………37
Figure. 11血小板貼附至fibrinogen過程Reln的影響…………………………… 38
Figure. 12血小板貼附至fibrinogen過程Reln對於血小板內F-actin結構的影響 39
Figure. 13血小板貼附至fibrinogen過程抑制Reln訊息傳遞後的影響………… 40
Figure. 14分析血小板內PKC活性受到Reln重組蛋白處理的變化…………… 41
Figure. 15分析血小板內Rac1活性受到Reln重組蛋白處理的變化…………… 42
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