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研究生:簡禎瑩
研究生(外文):Chien Chen-Ying
論文名稱:血纖維蛋白溶酶原片段K4418造成內皮細胞凋亡扮演的角色
論文名稱(外文):The role of plasminogen fragment K4418 in apoptosis of endothelial cells
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:慈濟大學
系所名稱:醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:中文
論文頁數:86
中文關鍵詞:血纖維蛋白溶酶原細胞凋亡
外文關鍵詞:plasminogenapoptosisFAKcaspasecalpain
相關次數:
  • 被引用被引用:0
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血管新生作用(angiogenesis),由舊有血管長出新血管的過程,是胚胎發育、傷口修復
及成人女性生理週期所需的生理過程。腫瘤生長或轉移依賴血管新生,因此抑制血管新
生可用於癌症治療。血纖維蛋白溶酶原(plasminogen)前四個kringle domain 的血管靜止
蛋白(angiostatin)能有效抑制血管新生。我們之前結果顯示不同的血纖維蛋白溶酶原片段
具有不同抑制血管新生的成效,而K4418 在抑制腫瘤生長成效上較K1-4 (angiostatin)為
佳。本實驗旨在找出K4418 引起細胞凋亡的機制並釐清參與作用的分子。重組蛋白K4418
以Pichia pastoris 系統表現再以DEAE 及Lysine Sepharose 純化。純化之K4418 會造成內
皮細胞凋亡呈一劑量效應。K4418 造成bFGF 引發之蛋白酪胺酸磷酸化的下降,尤以36
kDa~47 kDa 分子最為明顯。此現象會因給予酪胺酸去磷酸酶抑制物Na3VO4 而恢復。預
先處理Na3VO4 可抑制K4418 引起之細胞凋亡。偵測K4418 作用後酪胺酸去磷酸酶總活
性,結果顯示酪胺酸去磷酸酶活性在30 分鐘時下降,60 分鐘時有最高活性。之前結果
推測牛肺動脈內皮細胞integrin αvβ3 可能為K4418 的受體。FACScan 結果顯示人類臍靜
脈內皮細胞有integrin α1、α2、α3、αvβ3 和β1 的表達。酪胺酸去磷酸酶SHP-1,bFGF 引
起之增生訊息中的負調控者,會與integrin α3、β1、αv 和β3 結合。給予K4418 促使SHP-1
與β1、β3 integrins 間之結合增加。與β integrins 結合的focal adhesion kinase (FAK)對於細
胞貼附與生存是重要的。血管靜止蛋白會引起FAK 的持續活化及內皮細胞的凋亡。雖
然K4418 快速地引起FAK 的活化,然而在作用一小時後即造成FAK 的去磷酸化與降解。
K4418 作用兩小時會引發caspase 1、caspase 8 及calapin 的活化。而caspase 抑制物
(z-VAD-FMK 、z-DQMD-FMK) 、calpain 抑制物(ALLN) 及SHP-1 抑制物(sodium
stibogluconate)可抑制K4418 引起的FAK 去磷酸化與降解。根據這些結果,本研究推測
K4418 經由integrins 與內皮細胞接觸,引發細胞內酪胺酸去磷酸酶活性上升。SHP-1 或
是其他酪胺酸去磷酸酶在FAK 的失活上可能扮演重要角色。去磷酸化的FAK 進而被
K4418 活化之caspases 與calpain 降解,最終內皮細胞走向凋亡。
Angiogenesis, the process by which new blood vessels sprout from existing vessels to
vascularize tissues, is a necessary physiological process for embryonic development, wound
healing, and reproductive cycle in adult females. Tumor growth or metastasis is angiogenesis
dependent; therefore, antiangiogenesis can be applied for cancer therapy. Angiostatin, the first
four kringles domain of plasminogen, inhibits angiogenesis effectively. Our previous study
showed that various plasminogen fragments had different inhibitory effects on angiogenesis.
Especially, K4418 had better inhibitory effect on tumor growth than K1-4 (angiostatin). This
study was established to find out the apoptotic mechanism induced by K4418, and clarified the
molecules involved in the processes. Recombinant K4418 was expressed in Pichia pastoris
expression system and purified by anion exchange (DEAE) and Lysine Sepharose. Purified
K4418 could induce endothelial cell apoptosis in a dose-dependent manner. The bFGF-induced
total tyrosine phosphorylation was decreased by K4418, especially molecules between 36
kDa~47 kDa. The effect was rescued by the tyrosine phosphatase inhibitor Na3VO4.
Pretreatment with Na3VO4 could inhibit K4418-induced apoptosis. Detection of the total
tyrosine phosphatase activity after K4418 treatment showed that phospho-tyrosine phosphatase
activity was decreased at 30 min and had highest activity at 60 min. Previous data suggested
that integrin αvβ3 may be a receptor of K4418 in calf pulmonary artery endothelial cells
(CPAE). FACScan results showed that human umbilical vein endothelial cells (HUVEC)
expressed α1, α2, α3, αvβ3 and β1 integrins. Tyrosine phosphatase SHP-1, a negative regulator
of bFGF-induced proliferation signal, was associated with integrin α3, β1, αv and β3. After
K4418 treatment, SHP-1 associated with β1 and β3 integrins was increased. β integrins
associated focal adhesion kinase (FAK) is important in cell adhesion and survival. Angiostatin
induced FAK constitutive activation and endothelial cell apoptosis. Although K4418 rapidly
3
induced FAK activation, FAK was then dephosphorylated and degradated after 1 hr treatment.
Treatment with K4418 for 2 hr caused caspase 1, caspase 8 and calpain activation. Caspase
inhibitors (z-VAD-FMK and z-DQMD-FMK), calpain inhibitor (ALLN) and SHP-1 inhibitor
(sodium stibogluconate) could inhibit K4418-induced FAK dephosphorylation and degradation.
According to these results, this study suggested that K4418 interacted with endothelial cells
through integrins and increased total tyrosine phosphatase activity. SHP-1 or other tyrosine
phosphatase may play an important role in FAK inactivation. Dephosphorylated FAK was
further degraded by the caspases and calpain which were activated by K4418. Finally
endothelial cells underwent apoptosis.
中文摘要 1
英文摘要 2
誌謝 4
目錄 5
圖目錄 7
附錄目錄 8
緒論
1、血管新生作用與其調控 9
2、血管新生與腫瘤間的關係 11
3、血管靜止蛋白及其相近蛋白 12
4、血管靜止蛋白的作用機制 14
5、血管靜止蛋白與細胞凋亡 17
6、實驗目的 19
材料與方法
1、K4418 蛋白表現:
1-1、轉殖株小量蛋白質表現 20
1-2、蛋白質電泳 (protein electrophoresis) 21
1-3、西方點墨法 (Western blot) 23
1-4、轉殖株大量蛋白質的表現 25
1-5、重組蛋白質的純化 26
1-6、重組蛋白質的濃縮 27
1-7、蛋白質定量 27
6
2、內皮細胞繼代培養:
2-1、細胞培養 28
2-2、保存細胞 29
2-3、解凍細胞 30
2-4、細胞計數 30
3、重組蛋白K4418之活性分析:
3-1、細胞凋亡分析 31
3-2、細胞表面integrin表現分析 32
3-3、免疫沉澱法 32
3-4、Casein zymography 34
3-5、Cell adhesion assay 35
3-6、酪胺酸去磷酸酶活性分析(Tyrosine phosphatase activity assay) 36
4、統計及密度分析:
4-1、統計分析法 38
4-2、密度分析法 38
實驗結果與討論 39
參考文獻 55
圖 62
附錄 81
自述 86
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