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研究生:張淳淳
研究生(外文):Chun-chun Chang
論文名稱:血纖維蛋白溶酶原片段K4430造成內皮細胞凋亡過程中鈣離子之角色探討
論文名稱(外文):Role of Ca2+ in plasminogen fragement K4430 induced endothelial cell apoptosis
指導教授:林銘德林銘德引用關係
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:77
中文關鍵詞:細胞凋亡血纖維蛋白溶酶原鈣離子
外文關鍵詞:Ca2+plasminogen fragementapoptosis
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血管新生(Angiogenesis)是指從舊有血管新生出新血管的過程,在正常情況下胚胎發育、組織修補及女性生理週期等都受到血管新生的調控。許多研究發現,腫瘤的生長和轉移同時也伴隨著血管新生,因此抑制血管新生是近年來治療癌症的新方針。血管靜止蛋白(Angiostatin)是血纖維蛋白溶酶原(plasminogen)的一個片段蛋白,結構包含四個完整的kringle,它是一種能抑制血管新生的內生性蛋白。之前的研究發現,血纖維蛋白溶酶原片段K4430對內皮細胞造成凋亡的現象是藉由增加細胞內鈣離子濃度所引起。因此本篇論文將探討K4430造成內皮細胞鈣離子上升之作用機制。
K4430由酵母菌系統表現後,經由陰離子交換樹脂(DEAE)與親和性樹脂(lysine sepharose)純化。再以純化出來的K4430作用於牛肺動脈內皮細胞(CPAE),結果發現K4430引起的細胞凋亡呈現劑量效應。此外,K4430可以促使細胞內鈣離子濃度增加,且內鈣離子螯合劑 (BAPTA–AM)能夠有效抑制K4430所引起之細胞凋亡,而外鈣離子螯合劑(EGTA)則否。因此,由以上的結果說明K4430透過增加細胞內鈣離子濃度而造成內皮細胞凋亡,且增加的鈣離子是由細胞內釋放而來。由於細胞內鈣離子的恆定會透過粒線體和內質網的鈣離子通道做調控,因此使用數種針對粒線體和內質網上的鈣離子通道抑制劑,探討在K4430作用下細胞內增加的鈣離子來源。結果顯示K4430不會造成粒線體的鈣離子濃度下降,且使用粒線體的鈣離子通道抑制劑cyclosporine A以及CGP37157也不會抑制K4430造成細胞凋亡的現象。反之,K4430會引起內質網的鈣離子濃度下降,且使用內質網IP3 receptor的抑制劑2-APB、XesC及TMB-8都可以有效抑制K4430造成的細胞凋亡,而Dantrolene的抑制劑Ryandine則否。因此K4430造成內皮細胞凋亡是透過內質網上IP3 receptor釋放鈣離子至細胞內所引起。
由於細胞內IP3的來源主是要來自於活化的Phosphoinositide-specific phosphlipase C (PLC)會將phosphatidylinositol 4,5-bisphosphate (PIP2) 水解成 inositol 1,4,5-triphosphate (IP3) 和 diacylglycerol (DAG)。故接著探討K4430對內皮細胞造成凋亡的現象是否PLC也參與其中。結果發現在K4430作用下,細胞內PLCγ的磷酸化以及IP3濃度都有明顯的增加,且PLC的抑制劑-U73122可有效抑制K4430造成的內皮細胞IP3濃度增加以及細胞凋亡。從結果可以說明K4430會藉由活化PLCγ造成IP3的產生,進而引起IP3 receptor釋放鈣離子至細胞內造成細胞凋亡。
Angiogenesis, formation of new blood vessels from existing endothelium, occurs in embryonic development, tissue growth and female reproductive cycle. Tumor growth and metastasis are also angiogenesis-dependent, thus, anti-angiogenesis is a strategy for cancer therapy. Angiostatin, the first four kringles of plasminogen, is an endogenous inhibitor of angiogensis.
Our previous results revealed that plasminogen fragment K4430 caused calf pulmonary artery endothelial cells (CPAE) to undergo apoptosis in a dose-dependent manner. In this study, the result induced that K4430 caused an increase in intracellular Ca2+ level, and intracellular Ca2+ chelator, BAPTA-AM, could significantly inhibit K4430-induced cell apoptosis. Mitochondrial and endoplasmic reticulum (ER) Ca2+ channels have been involved in the regulation of cytosolic Ca2+ level. Several of Ca2+ channel blockers of both channels were used in this study to evaluate their role in K4430-induced apoptosis. The results showed K4430 could not induce mitochondrial Ca2+ decrease, and mitochondria Ca2+ release blocker, cyclosporine A and CGP37157 had no effect on K4430-induced apoptosis. However, K4430 could induce ER Ca2+ decrease, and the blockers of IP-3 receptor, 2-APB , Xesnospogin C and TMB-8 could inhibit K4430 induced-apoptosis. These results demonstrated that Ca2+ release from ER through IP3 receptor into cytosol might be necessary for K4430 induced-apoptosis.
PLC catalyzed the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) to inositol 1,4,5-triphosphate (IP3) and diacylglycerol (DAG). Binding of IP3 to IP3 receptor could promote the release of Ca2+ from ER. Our results showed that K4430 enhanced the phosphorylation of PLC γ and the production of IP3 and the PLC inhibitor, U73122, could inhibit K4430-induced level of IP3 and apoptosis. These results suggest that K4430 induced-apoptosis is mediated by the intracellular Ca2+ increase from ER, which is the result of the phosphorylation of PLC γ and the production of IP3.
目錄 1
中文摘要 4
Abstract 5
緒論 6
血管新生作用與其調控: 6
血管靜止蛋白(angiostatin): 7
血管靜止蛋白抗血管新生作用的機制: 9
鈣離子與細胞凋亡的關係 10
研究動機 12
材料與方法 14
轉殖株小量蛋白質的表現 14
轉殖株蛋白質的表現 15
重組蛋白質的純化 16
重組蛋白的濃縮 17
蛋白質定量 18
蛋白質電泳 (protein electrophoresis) 19
西方點墨法 (western blot) 21
細胞繼代培養 24
保存細胞 25
解凍細胞 26
細胞計數 27
細胞凋亡分析 27
測量細胞質內鈣離子濃度 28
測量粒線體中鈣離子濃度 29
測量內質網鈣離子濃度 30
抑制鈣離釋放之抑制劑 31
偵側PLC-γ的磷酸化 32
測量細胞內IP3的變化 35
實驗結果 38
酵母菌轉殖株的篩選與小量表現 38
大量重組蛋白的純化 38
K4430引起內皮細胞凋亡 39
K4430引起內皮細胞內鈣離子的增加 39
鈣離子螯合劑可以抑制K4430造成的內皮細胞凋亡 40
K4430引起內皮細胞凋亡現象中粒線體扮演的角色 40
K4430引起內皮細胞凋亡現象中內質網扮演的角色 41
K4430引起內皮細胞內鈣離子增加現象中PLCγ扮演的角色 42
討論 44
參考文獻 50
結果圖 56
附錄I:儀器 72
附錄II:縮寫簡索表 74
附錄III:Vector Map of pPICZαA 75
附錄IV:mouse plasminogen的胺基酸序列 76
自述 77
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