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研究生:張文馨
研究生(外文):Wen-Hsin Chang
論文名稱:血纖維蛋白溶酶原片段K4(20-436)和K4(20-448)的抗血管新生活性
論文名稱(外文):The Anti-angiogenic Activities of Plasminogen Fragments K4(20-436) and K4(20-448)
指導教授:林銘德林銘德引用關係
指導教授(外文):Ming-T, Lin
學位類別:碩士
校院名稱:國立成功大學
系所名稱:生物化學研究所
學門:生命科學學門
學類:生物化學學類
論文種類:學術論文
論文出版年:2003
畢業學年度:91
語文別:英文
論文頁數:74
中文關鍵詞:血管新生血管靜止蛋白細胞凋亡內皮細胞
外文關鍵詞:angiogenesisangiostatinapoptosisendothelial cell
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摘要
血管新生(angiogenesis),是指從已存在的血管新生出微血管的過程,在正常生理中發生於組織生長、損傷修復以及女性的生理週期。由於血管新生也是腫瘤生長與轉移所必須的,因此抑制血管新生是治療癌症的策略之一。血管靜止蛋白(angiostatin),是血纖維蛋白溶酶原(plasminogen)前四個kringle的一個片段蛋白,它是一種能抑制血管新生的內生性蛋白。本實驗室之前研究發現兩個血纖維蛋白溶酶原片段蛋白(K420-436與K420-448),這兩個蛋白對內皮細胞增生與移動的抑制效果比血管靜止蛋白(K420-461)還顯著。本實驗的目的便是更進一步的闡明這兩個片段蛋白抑制血管新生作用的機制。實驗所使用的血纖維蛋白溶酶原片段蛋白,是以酵母菌表現系統表現,經由陰離子交換樹脂(DEAE)與親和性樹脂(lysine sepharose)純化後所得。由於糖化的結果,表現蛋白的分子量比預估的大,而且這些糖類大多由mannose所構成。我們接著研究血纖維蛋白溶酶原片段蛋白的接受器以企圖了解它們的作用。ATP synthase,被認為是人類臍靜脈內皮細胞(HUVEC)表面上的血管靜止蛋白接受器,但是它卻不存在於牛肺動脈內皮細胞(CPAE)的表面。由於以血纖維蛋白溶酶原片段蛋白處理細胞之後,細胞會開始懸浮,因此我們猜測integrins是這些蛋白的接受器。在檢視integrin於細胞表面的表現量發現,除了 a2之外,a1、 b1 以及avb3皆會表現。avb3,與含有RGD (Arg-Gly-Asp) 序列的蛋白有很強的親和性,而且它是血管新生過程中一個十分重要的蛋白。因此我們更進一步的探討avb3是否可與血纖維蛋白溶酶原片段蛋白結合。細胞若先以avb3抗體或RGD peptides處理,則無法再與固定的片段蛋白黏著,顯示出血纖維蛋白溶酶原片段蛋白與牛肺動脈內皮細胞的結合是經由avb3,而且儘管血纖維蛋白溶酶片段蛋白沒有RGD的序列,這種結合仍然顯示出是RGD-dependent。以血纖維蛋白溶酶原片段蛋白處理牛肺動脈內皮細胞後,發現這些貼附的細胞開始懸浮以及走向細胞凋亡(apoptosis; anoikis),但是對於非內皮細胞(A549)則沒有任何影響,而且片段蛋白K420-436與K420-448促使細胞凋亡的能力,比血管靜止蛋白(K420-461)還顯著。若是細胞在處理血纖維蛋白溶酶原片段蛋白前先以廣效性的caspase抑制劑(zVAD-FMK)處理,細胞凋亡的現象便可以完全地被抑制住。這顯示出caspase在血纖維蛋白溶酶原片段蛋白引發的細胞凋亡中,扮演著十分重要的角色。而在細胞凋亡過程中,caspase 9 與caspase 3會被活化而且細胞質中的cytochrome c也會增加。
Angiogenesis, the sprouting of new capillaries from established vessels, occurs physiologically during tissue growth, wound healing, and female reproductive cycle. Because tumor growth and metastasis are also angiogenesis-dependent, anti-angiogenesis is a strategy for cancer therapy. Angiostatin, the first four kringles of plasminogen, is an endogenous inhibitor of angiogenesis. Pervious studies in our laboratory found two plasminogen fragments, K420-436 and K420-448, which exhibited more remarkable anti-proliferative and anti-migratory activities than K420-461 (angiostatin). The purpose of this study was to further elucidate the mechanism of how they inhibit angiogenesis. Plasminogen fragments were expressed in Pichia pastoris and purified by anion exchange (DEAE) and lysine sepharose. Molecular weights of these proteins were larger than predict ones due to glycosylation and most of these saccharides attached to plasminogen fragments were mannose. The receptors for these proteins were studied in attempt to realize their action. ATP synthase, which was believed to be a receptor for angiostatin on the surface of human umbilical vein endothelial cells (HUVECs), did not exist on the calf pulmonary artery endothelial cell (CPAE) surface. Because the morphological changes, cell rounded and detached, were observed after treatment, integrins were suspected to be receptors for plasminogen fragments. The expression of a1, a2, b1 and avb3 integrins on CPAE cells was examined and all of them were constitutively expressed except a2. Integrin avb3, which had a high affinity for RGD (Arg-Gly-Asp) sequence-containing proteins, was a fundamental protein in angiogenesis so it was further studied whether they bound to plasminogen fragments. Cells pre-incubated with anti-avb3 antibody or RGD peptides could not adhere to immobilized plasminogen fragments anymore. The results indicated plasminogen fragments were bound to CPAE cells through avb3 in an RGD-dependent manner despite that there is no RGD sequence in plasminogen fragments. Following treatment with plasminogen fragments, CPAE cells underwent apoptosis (anoikis) but not non-endothelial cells (A549) and the apoptotic effects of K420-436 and K420-448 were much higher than K420-461. The apoptosis of CPAE cells could completely be abolished by pre-treatment with a broad caspase inhibitor (zVAD-FMK) indicating caspases played a fundamental role in the plasminogen fragments- induced apoptosis. In the apoptotic pathway, caspase 9 and caspase 3 were greatly activated and cytosolic levels of cytochrome c increased.
Chinese Abstract 1
Abstract 3
Acknowledgements 5
Contents 6
Index of figures 8
Index of appendixes 10
Introduction 11
Process and Regulation of Angiogenesis 11
Discovery and Characteristic of Angiostatin 12
Mechanism of Action 14
Angiogenesis and Apoptosis 15
Rationale for This Study 16
Materials and Methods 18
Angiostatin Production and Purification 18
Electrophoresis and Western Blotting 19
Glycoprotein Detection 21
Deglycosylation 22
Cell Culture 23
Cell Surface Staining 24
Cell Adhesion Assay 24
Hoechst DNA Assay 25
Apoptosis Analysis by Propidium Iodide Assay 26
Assessment of Cytochrome C Release 27
Measurement of Caspase Activity 28
Results 29
Purification of Plasminogen Fragments 29
Deglycosylation of Plasminogen Fragments 29
Adhesion of CPAE cells to Plasminogen Fragments 30
Not Expression of ATP Synthase on CPAE Cell Surface 30
Integrin avb3-mediated CPAE cell adhesion 30
Apoptosis Induced by Plasminogen Fragments 31
Involvement of mitochondria in apoptosis 32
Caspases activity induced by plasminogen fragments 32
Discussion 33
References 39
Figures 48
Appendix 67
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