臺灣博碩士論文加值系統

CHOαIIbβ3 細胞在細胞膜上表現外生性human integrinαIIbβ3，會藉由具有fibrinogens or disintegrins 基質處理後，活化並產生細胞內鈣離子的濃度變化。在這些細胞中，發現細胞質內具有囊泡狀的結構，為sodium-proton exchanger NHE1、sodium-calcium exchanger NCX1 與integrinαIIbβ3 發生反應從細胞質移動到細胞膜上的情況。利用FRET-FLIM 的技術測量螢光生命期，卻可觀測到這些分子間的交互反應。進一步的，我們發現integrinαIIbβ3 與這些ion exchangers 彼此間的作用會形成functional complex，並且開啟細胞內鈣離子的變動，而這些機制由lipid microdomain 或lipid rafts 參與。

Chinese hamster ovary cells expressing exogenous human integrinαIIbβ3 on their plasma membrane (CHOαIIbβ3) exhibited active calcium oscillations when plated on substrates coated with fibrinogens or disintegrins. In these
cells, profound targeting of sodium-proton exchanger NHE1 and sodium-calcium exchanger NCX1 from intracellular vesicles to the plasma membrane was noticed, where they co-localized with integrins. Such molecular interactions could be clearly demonstrated using FRET-FLIM technique based on fluorescence lifetime measurements. Further more, targeting and interaction of the ion exchangers with integrinαIIbβ3, and their functional coupling to initiate calcium influx, were dependent on lipid microdomain, or lipid rafts.

一. 序論
1.1 Calcium-induced calcium release（CICR）的生理意義
1.2 integrin 與其下游的訊號傳遞
1.3 integrinαIIbβ3 在血小板內扮演的角色
1.4 細胞外基質Fibrinogen 與蛇毒蛋白Rhodostomin
1.5 NHE1 與NCX1 在細胞中參與的機制
1.6 Lipid raft 對於細胞訊息的調控
1.7 介紹螢光顯微術
1.7.1 螢光的原理
1.7.2 螢光共振能量轉移（Fluorescence resonance energy transfer，FRET）
1.7.3 雙光子激發顯微技術（Two-photon excitation microscopy）
1.7.4 螢光生命期影像顯微鏡（Fluorescence lifetime image
microscopy，FLIM）
1.7.5 單光子計數系統（Time–correlated single photon counting，TCSPC）
二. 材料與方法
2.1 材料
2.2 方法
2.3 儀器簡介
2.4 數據分析與影像擷取
三. 結果
1. CHOαIIbβ3 細胞吸附介質後觀測NHE1、NCX1、integrinαIIbβ3 蛋白在細胞裡的交互反應
2. CHOαIIbβ3 細胞吸附介質後觀測NHE1、NCX1 蛋白在細胞裡的交互反應
3. 觀測CHOαIIbβ3 細胞與coating beads 接觸後NHE1、NCX1、
integrinαIIbβ3 蛋白在細胞裡的交互反應
4. Rhodostomin beads 與表現NHE1-EGFP 與integrinβ3-mRFP 螢光蛋白的細胞之反應
5. 利用drug treatment 研究CHOαIIbβ3 活細胞內NHE1 integrinαIIbβ3 蛋白參與的機制
6. CHOαIIbβ3 細胞吸附介質後觀測NHE1、NCX1、caveolin 蛋白在細胞膜的交互反應
7. Non raft marker CD45 不會與NHE1、NCX1 蛋白發生交互反應
四. 討論
1. 探討NHE1、NCX1、integrinαIIbβ3 蛋白在細胞裡的交互反應
2. 探討integrinαIIbβ3 蛋白與NHE1、NCX1 在細胞裡的參與機制
3. Lipid raft 影響NHE1、NCX1 蛋白在細胞膜上的組裝
4. 探討FRET-FLIM 技術對於蛋白質交互反應的偵測
五. 參考文獻
六. 圖表

1. Becker, W., A. Bergmann, and C. Biskup, Multispectral fluorescence lifetime imaging by TCSPC. Microsc Res Tech, 2007. 70(5): p. 403-9.
2. Becker, W., et al., Fluorescence lifetime images and correlation spectra obtained by multidimensional time-correlated single photon counting.Microsc Res Tech, 2006. 69(3): p. 186-95.
3. Becker, W., et al., Fluorescence lifetime imaging by time-correlated single-photon counting. Microsc Res Tech, 2004. 63(1): p. 58-66.
4. Berridge, M.J., Cardiac calcium signalling. Biochem Soc Trans, 2003. 31(Pt5): p. 930-3.
5. Berridge, M.J., M.D. Bootman, and H.L. Roderick, Calcium signalling:dynamics, homeostasis and remodelling. Nat Rev Mol Cell Biol, 2003. 4(7): p.517-29.
6. Buensuceso, C.S., et al., Regulation of outside-in signaling in platelets by integrin-associated protein kinase C beta. J Biol Chem, 2005. 280(1): p.644-53.
7. Blaustein MP, Lederer WJ. Sodium/calcium exchange: its physiological implications. Physiol Rev. 1999 Jul;79(3):763-854. Review.8. Chang, M.C., et al., The morphologic change of endothelial cells by ancrod-generated fibrin is triggered by alpha v beta 3 integrin binding and the subsequent activation of a G-protein coupled phospholipase C. Biochim Biophys Acta, 1995. 1269(2): p. 115-21.
9. Chao, C.C. and S.L. Huang, Inhibition of UV-damaged DNA recognition activity in HeLa cells by calcium ionophore A23187: potential involvement of cytosolic proteins. Biochem Biophys Res Commun, 1993. 193(2): p.764-70.
10. Chao, W., and Olson, M. S. (1993). Platelet-activating factor: receptors and signal transduction. Biochem J 292 (Pt 3), 617-629.11. Clark E.A. and Brugge J.S., Integrins and signal transduction pathways: the road taken. Science 268: 233–239,1995.
12. Dedhar, S. and G.E. Hannigan, Integrin cytoplasmic interactions and bidirectional transmembrane signalling. Curr Opin Cell Biol, 1996. 8(5): p.657-69.
13. Denker, S. P., & Barber, D. L. (2002). Cell migration requires both ion translocation and cytoskeletal anchoring by the Na+/H+ exchanger NHE1.The Journal of Cell Biology, 159, 1087–1096.
14. Der-Shan Sun, Szecheng J. Lo, Chi-Hung Lin, Mei-Shiuan Yu, Ching-Yi Huang,Yao-Fong Chen & Hsin-Hou Chang (2005)., Calcium oscillation and phosphatidylinositol 3-kinase positively regulate integrinαIIbβ3-mediated outside-in signaling. J. Biom .Sci 12：321-333.
15. DiPolo, R. and L. Beauge, Sodium/calcium exchanger: influence of metabolic regulation on ion carrier interactions. Physiol Rev, 2006. 86(1): p.155-203.
16. Elangovan, M., et al., Characterization of one- and two-photon excitation fluorescence resonance energy transfer microscopy. Methods, 2003. 29(1): p.58-73.
17. Gebreselassie D, Rajarathnam K, Fliegel L. (1998) Expression, purification,and characterization of the carboxyl-terminal region of the Na+/H+ exchanger. Biochem Cell Biol. 76(5):837-42
18. Giancotti, F.G. and E. Ruoslahti, Integrin signaling. Science, 1999. 285(5430):p. 1028-32.
19. Guilherme, A., et al., A novel phosphorylated lipid counteracts activation of the renal plasma membrane (Ca2+ + Mg2+)ATPase by endogenous phosphatidylinositol-4-phosphate. Biosci Rep, 1998. 18(2): p. 79-89.
20. Keularts, I.M., et al., alpha(2A)-adrenergic receptor stimulation potentiates calcium release in platelets by modulating cAMP levels. J Biol Chem, 2000.275(3): p. 1763-72.