臺灣博碩士論文加值系統

1. 嗜中性白血球為身體免疫系統先天和體液性中扮演舉足輕重的作用細胞，且為對抗微生物的第一防線。然而，經由發炎及自體免疫疾病中觀察到對細菌的謀殺及對宿主組織破壞能力，暗示著嗜中性白血球具有一體兩面的作用；一體兩面的能力是藉由NAD(P)H 氧化酶聚合與轉位，代謝O2產物而達其結果，其調節受到精準調控。如何有效抑制O2產物對宿主組織破壞，遂成為現今重要研究題目之一。
Caffeic acid phenethyl ester (CAPE) 是由蜂膠中分離出的一種成分，具有抗氧化以及抗發炎的作用。但是，在人類嗜中性白血球中CAPE如何抑制細菌蛋白合成產物N-formylpeptides (fMLP) 刺激所產生的超氧自由基釋放，其藥物的作用機制仍不清楚。是否這一系列化合物皆具有相同藥效?
本研究先篩選這類藥物抑制fMLP活化嗜中性白血球超氧自由基釋放比率，結果顯示CAPE抑制較為顯著。CAPE 對於fMLP及PMA刺激超氧自由基釋放的IC50分別為7.40 ± 4.10M及6.55 ± 1.60M；相對CAPE在無細胞的系統中也具有清除自由基的能力，IC50為3.99 ± 1.32M。 CAPE亦會抑制xanthine oxidase的活性，IC50為45.79 ± 11.79M。
fMLP活化嗜中性白血球產生超氧自由基的機制可經鈣離子釋放和PKC及NAD(P)H氧化酶轉位而達成；闡明CAPE抑制路徑，進一步觀察其細胞內鈣離子、PKC及NAD(P)H氧化酶表現差異。結果得知，CAPE可抑制fMLP及PMA刺激NAD(P)H 氧化酶活化及抑制fMLP及PMA所引起的cPKC 及p47phox translocation；但對於fMLP刺激的細胞內鈣離子不具有抑制作用。
總結，CAPE可能是經由抑制cPKC 及p47phox translocation，而抑制NAD(P)H氧化酶活化，遂抑制fMLP及PMA所造成的超氧自由基釋放。
2.在後基因體世代中，同時運用基因體學、蛋白質體學及生物資訊學三種科學來分析生物系統中基因及蛋白質的表現，即成為目前系統生物學最先進的研究技術。無論如何，基因體學不能預測蛋白質轉譯修飾之構形，蛋白質體學卻能提供此種分析。何謂蛋白質體學？蛋白質體學結合了複雜的技術，包括二維電泳分析、膠體影像分析、質譜儀胜肽指紋、氨基酸序列定序及生物資訊應用來鑑定蛋白質。在本文研究人類嗜中性白血球、人類及兔子血小板以及高血壓型及正常型大鼠平滑肌細胞的二維電泳圖譜。在嗜中性白血球，大多分析出與細胞骨架相關的蛋白質，其與嗜中性白血球所調控的作用為吸附作用。在血小板中，大多分析出與血液凝集及細胞骨架相關的蛋白質。而在平滑肌細胞中主要是與肌肉收縮及生長相關的蛋白質。本文的研究目的，是希望建立完整的蛋白質資料庫，以利於疾病治療以及新藥開發的研究。

1.The neutrophil is body’s first line of defense against microorganisms and a critical effector cell in both innate and humoral immunity. However, the capacity for bacterial killing carriers with it an implicity for host tissue destruction, as observed in inflammatory and autoimmune disease. Neutrophils produce potentially dangerous metabolites from superoxide in the defense against microorganisms. Alternatively, caffeic acid phenethyl ester (CAPE) was isolated from propolis that has many biological and pharmacological activities including anti-oxidation and anti-inflammatory. Although CAPE has antioxidant effects, its mechanism on the human neutrophils after fMLP treatment has not been investigated to date.
Here we show that CAPE can inhibited fMLP and PMA stimulated human neutrophils superoxide production (IC50 : 7.40 ± 4.10M and 6.55 ± 1.60M). In xanthine/xanthine oxidase system, CAPE possessed superoxide scanverger effect (IC50 : 3.99 ± 1.32M) and inhibited xanthine oxidase activity (IC50 : 45.79 ± 11.79M).
To further determine the mechanism of CAPE-induced inhibitory effect on the activation of fMLP-treated neutrophils. We measured the intracellular calcium level and NAD(P)H oxidase activity. Here we show that CAPE can inhibit fMLP and PMA from stimulating NAD(P)H oxidase activity, but not to inhibit fMLP from increasing intracellular calcium. However CAPE can inhibit fMLP which courses PKC translocation.
Taken together, these data suggest that CAPE inhibits the fMLP-induced neutrophil activation maybe through the inhibition of cPKC and NAD(P)H oxidase activity leading to the reduction of ROS.
2.In post genomic era, simultaneous with genomics, proteomics and bio-informatics to analyze an organisms that gene and protein expression, to investigate systematic biology. However, considering splice variants and post-translational modification of protein, the number of all structurally and functionally will probable exceed 30,000 ~ 40,000 by at least 10 ~ 200 fold. Proteomics has the power to provide comprehensive structural and quantitative information on all these proteins. Proteomics uses a combination of sophisticated techniques including two-dimensional gel electrophoresis, image analysis, mass spectrometry, amino acid sequencing, and bio-informatics to characterize proteins. In this article, the development of a two-dimensional map of human neutrophil, human platelet, rabbit platelet, spontaneously hypertension (SHR) rat vascular smooth muscle cell and sprague-dawley (SD) rat vascular smooth muscle cell. In neutrophil section, a large number of cytoskeleton proteins were detected, which related to neutrophil function such as adhesion. In platelet section, a large number of blood-coagulant and cytoskeleton proteins were detected. In vascular smooth muscle cell section, a large number of muscle concentrate and growth proteins were detected. The aim of this project is to build up a complete database of such proteins, to be utilized for future studies where is used as a disease or drug treatment marker.

1.
縮 寫 表 - 1 -
中文摘要 - 3 -
英文摘要 - 4 -
緒論 - 5 -
研究動機 - 12 -
實驗材料 - 13 -
實驗方法 - 14 -
結果 - 19 -
討論 - 23 -
參考文獻 - 39 -
2.
縮寫表 - 44 -
中文摘要 - 45 -
英文摘要 - 46 -
緒論 - 47 -
研究動機 - 53 -
實驗材料 - 55 -
實驗方法 - 56 -
結果 - 61 -
討論 - 67 -
參考文獻 - 105 -

1.
Ali, H., Richardson, R.M., Haribabu, B., and Snyderman, R. (1999) Chemoattractant receptor cross-desensitization. J. Biol. Chem. 274:6027-6030
Allen, R.G.. and Tresini, M. (1999) Oxidative stress and gene regulation. Free Radaci. Biol. Med. 28:463-499
Babior, B.M. (1999) NADPH oxidase : an update. Blood 93:1464-1476
Balasubramanian, N., Advani, S. H. and Zingde, S.M. (1998) Protein kinase C isoforms in normal and chronic myeloid leukemic neutrophils. Distinct signal for PKC a by immunodetection on PVDF membrane, decreased expression of PKC d in leukemic neutrophils. Leuk. Res. 22: 597-604
Balasubramanian, N., Advani, S. H. and Zingde, S.M. (2002) Protin kinase C isoforms in normal and leukemic neutrophils: altered levels in leukemic neutrophils and changes during myeloid maturation in chronic myeloid leukemia. Leuk. Res. 26: 67-81
Bergendi, L., Benes, L., Durackova, Z., Ferencik, M. (1999) Chemistry, physiology and pathology of free radicals. Life Sci. 65, 1865-1874
Casabona, G. (1997) Intacellular signal modulation : a pivotal role for protein kinase C. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 21: 407-425
Chen, Q., Powell, D.W., Rane, M.J., Singh, S., Butt, W., Klein, J.B., and McLeish, K.R. (2003) Akt phosphorylates p47phox and mediates respiratory burst activity in human neutrophils. J. Immunol.170:5320-5308
Chiao, C., Carothers, A. M., Grunberger, D., Solomon, G., Preston, G. A., Barrett, J. C. (1995) Apoptosis and altered redox state induced by caffeic acid phenethyl ester (CAPE) in transformed rat fibroblast cells. Cancer Res. 55, 3576-3583
Cui, X. L. and Douglas, J. G., (1997) Arachidonic acid activates c-jun N-terminial kinase through NADPH oxidase in rabbit proximal tubular epithelial cells. Proc. Natl. Acad. Sci. USA 94 : 3771-3776
Dang, P.M., Rais, S.A., Hakim, J., and Perianin, A, (1995). Redistribution of protein kinase C isoforms in human neutrophils stimulated by formyl peptide and phorbol myristats acetate. Biochem. Biophys. Res. Commun. 2:664-672
Dillon, S.B., Murray, J.J., Uhing, R.J., and Snyderman, R. (1987) Regulation of inositol phospholipid and inositol phosphate metabolism in chemoattractant-activated human polymorphonuclear leukocytes. J Cell Biochem. 35:345-59.
Escobar, J.A., Rubio, M.A., and Lissi, E.A. (1996) SOD and catalase inactivation by singlet oxygen and peroxyl radicals. Free.Radi.Bio.Med. 20:285-290
Forman, H.J., and Thomas, M.J. (1986) Oxidant production and bactericidal activity in phagocytes. Annu.Rev.Physiol 48:669-675
Frenkel, K., Wei, H., Bhimani, R.S., Ye, J.J., Zadunaisky, J.A., Huang, M.T., Ferraro, T., Conney, A.H., and Grunberger, D. (1993) Inhibition of tumor promoter-mediated processes in mouse skin and bovine lens by caffeic acid phenethyl ester. Cancer Res. 53:1255-1261
Griendling, K.K., Minieri, C.A., Ollerenshaw, J.D., and Alexander, R.W. (1994) Angiotensin Ⅱ stimulates NADP and NADPH oxidase activity in cultured vascular smooth muscle cells. Circ Res. 74:1141-1148
Grunberger, D., Banerjee, R., Eisinger, K., Oltz, E.M., Efros, L., Caldwell, M., Estevez, V., and Nakanishi, K.(1988) Preferential cytotoxicity on tumor cells by caffeic acid phenethyl ester isolated from propolis. Experientia 44:230-2
Hogg, J.C. (1987) Neutrophil kinetics and lung injury. Physiol.Rev. 67:1249
Jaiswal, A.K., Venugopal, R., Mucha, J., Carothers, A.M., and Grungerger, D. (1997) Caffeic acid phenethyl ester stimulates human antioxidant response element-mediated expression of the NAD(P)H : Quinone Oxidoreductase (NQO1) gene. Cancer Res. 57:440-446
Jones, R.D., Hancock, J.T., and Morice, A.H. (2000) NADPH oxidase : a universal oxygen sensor? Free.Radi.Bio.Med. 29:416-424
Knight, J.A. (2000) Review:Free radicals, antioxidants, and the immune system. Annals of Clinical Laboratory Science 30:145-159
Kowaltowaki, A.J., and Vercesi, A.E. (1998) Mitochondrial damage induced by conditions of oxidative stress. Free.Radi.Bio.Med. 26:463-471
Liochev, S.I., and Fridovich, I. (1998) Lucigenin as mediator of superoxide production: revisited. Free.Radi.Bio.Med. 25:926-928
Ma, H.T., Patterson, R.L., vanRossum, D.B., Birnbaumer, L., Mikoshiba, K., and Gill, D.L. (2000) Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels. Science 287:1647-1651
Marcucci, M.C., Bankova, V., Christov, R., Kujumgiev, A., and Popov, S. (1995) Chemical composition and antibacterial activity of Brazilian propolis. Zeitschrift fur Naturforschung - Section C - Biosciences. 50:167-72
Markert, M., Andrews, P.C., and Babior, B.M. (1979) Measurement of O2- production by human neutrophils. The preparation and assay of NADPH oxidase containing particles from human neutrophils. J. Biol. Chem. 254:358-369
Merritt, J.E., Mccarthy, S.A., Davies, M.P.A., and Moores, K.E. (1990) Use of (fluo-3/AM) to measure cytosolic Ca2+ in platelets and neutrpophils. Biochem J. 269;513-519
Pongracz, J. and Lord, J. M. (1998) Superoxide production in human neutropkils: Evdence for signal redundancy and involvement of more than one PKC isoenyme. Biochemicl and Biophysical research communications 247:624-629
Postma, B., Poppelier, M.J., Van Galen, J.C., Prossnitz, E.R., Van Strijp, J.A., De Haas, C.J., and Van Kessel, K.P. (2004) Chemotaxis Inhibitory Protein of Staphylococcus aureus Binds Specifically to the C5a and Formylated Peptide Receptor. J Immunol. 172:6994-7001.
Russo, A., Longo, R., and Vanella, A. (2002) Antioxidant activity of propolis: role of caffeic acid phenethyl ester and galangin. Fitoterapia suppl. 1: S21-S29
Schnabel, P. and Bohm, M. (1996) Heterotrimeric G protein in heart disease. Cell Signal 8: 413-423.
Selloum, L., Reichl, S., Muller, M., Sebihi, L., and Arnhold, J. (2001) Effects of flavonols on the generation of superoxide anion radicals by xanthine oxidase and stimulated neutrophils. Arch. Biochem. Biophy. 395, 49-56
Sergeant, S. and McPhail, L.C. (1997) Opsonized zymosan stimulates the redistribution of protein kinase C isoforms in human neutrophils. J. Immunol. 159: 2877-2885
Shen, Y.C., Chiou, W.F., Chou, Y.C., and Chen, C.F. (2003) Mechanisms in mediating the anti-inflammatory effects of baicalin and baicalein in human leukocytes. Eur. J. Pharmacol. 465:171-181
Shieh, D. E., Liu, L. T., and Lin, C. C. (2000) Antioxidant and free radical scavenging effects of baicalein, baicalin and wogonin. Anticancer Res. 20, 2861-2866.
Siomboing, X., Gressier, B., Dine, T., Brunet, C., Luyckx, M., Cazin, M. and Cazin, J. C. (2001) Investigation of the inhibitory effects of chelerythrine chloride on the translocation of the protein kinase C βⅠ , βⅡ , ζ in human neutrophils. IL Farmaco 56 : 859-865
Song, Y. S., Park, E. H., Hur, G. M., Ryu, Y. S., Lee, Y. S., Lee, J. Y., Kim, Y. M., and Jin, C. B. (2002) Caffeic acid phenethyl ester inhibits nitric oxide synthase gene expression and enzyme activity. Cancer Lett. 175, 53-61.
Spitz, D. R., and Oberley, L. W. (1989) An assay for superoxide dismutase activity in mammalian tissue homogenates. Anal. Biochem. 179, 8-18.
Stell, H.C., and Anderson, R. (2002) Dissociation of the PAF-receptor from NADPH oxidase and adenylate cyclase in human neutrophils results in accelerated influx and delayed clearance of cytosolic calcium. Br. J. Pharmacol. 136:81-89.
Sud'ina, G.F., Mirzoeva, O.K., Pushkareva, M.A., Sumbatyan, N.V., and Varfolomeev, S.D. (1993) Caffeic acid phenethyl ester as a lipoxygenase inhibitor with antioxidant properties. FEBS Lett 329:21-24
Su, Z.Z., Lin, J., Grunberger, D., and Fisher, P.B. (1994) Growth suppression and toxixity induced by caffeic acid phenethyl ester (CAPE) in type 5 adenovirus-transformed rat embryo cells correlate directly with transformation progression. Cancer Res.54:1865-1870
Usia, T., Banskota, A. H., Tezuka, Y., Midorikawa, K., Matsushige, K., and Kadota, S. (2002) Constituents of Chinese propolis and their antiproliferative activities. J. Nat. Prod. 65, 673-676.
Watabe, M., Hishikawa, K., Takayanagi, A., Shimuzu, N., and Nakaki, T. (2004) Caffeic acid phenethyl ester induces apoptosis by inhibition of NFB and activation of Fas in human breast cancer MCF-7 cells. J. Biol. Chem. 279, 6017-6026.
Wolin, M.S., Gupte, S.A. and Oeckler, R.A. (2002) Superoxide in the vascular system. J.Vasc.Res. 39:191-207
Yamamori, T., Inanami, O., Nagahata, H., and Kuwabara, M. (2004) Phosphoinositide 3-kinase regulates the phosphorylation of NADPH oxidase component p47phox by controlling cPKC/PKCdelta but not Akt. Biochem Biophys Res Commun. 316:720-30
2.
Aebersold, R., and Goodlent, D.R. (2001) Mass spectrometry proteomics. Chem.Rev. 101:269-295
Banks, R.E., Dunn, M.J., Hochstrasser, D.F.,Sanchez, J.C., Blackstock, W., Pappin, D.J. and Selby, P.J. (2000) Proteomics: new perspectives, new biomedical opportunities. Lancet 356:1749-1756
Blackstock, W. P. (2000) Trend in automation and mass spectrometry for proteomics. A Trends Guide 7:12-17
Burnette, W.N. (1981) "Western blotting": electrophoretic transfer of proteins from sodium dodecyl sulfate--polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A. Anal Biochem. 112:195-203.
Celis, J.E., Ostergaard, M., Rasmussen, H.H., Gromov, P., Gromova, I., Varmark, H., Palsdottir, H., Magnusson, N., Andersen, I., Basse, B., Lauridsen, J.B., Ratz, G., Wolf, H., Orntoft, T.F., Celis, P., and Celis, A. (1999) A comprehensive protein resource for the study of bladder cancer: http://biobase.dk/cgi-bin/celis. Electrophoresis 20:300-309
Chambers, G., Lawrie, L., Cash, P., and Murray, G.I. (2000) Proteomics:a new approach to the study of disease. J.Pathol. 192:280-288
Charakida, M.C., Tousoulis, D., Stefanadis, C., and Toutouzas, P. (2003) The role of platelet glycoprotein Ib and IIb polymorphism in coronary artery diaease. Hellenic J. Cardiol. 44:43-48
Clements, M.K., Siemsen, D.W., Swain, S.D., Hanson, A.J., Nelson-Overton, L.K., Rohn, T.T., Quinn, M.T. (2003) Inhibition of actin polymerization by peroxynitrite modulates neutrophil functional responses. J. Leukoc. Biol. 73:344-355
Craeford, M.E., Cusick, M.E., and Garrel, J.I. (2000) Databases and knowledge resourses for proteomics research. A Trends Guide 7:17-21
Djafarzadeh, S., and Niggli, V. (1997) Signaling pathways involved in dephosphorylation and localization of the actin-binding protein cofilin in stimulated human neutrophils. Exp Cell Res. 236:427-35.
Görg, A., Postel, W., Weser, J., Patutschnick, W., and Cleve, H. (1985) Improved resolution of PI (alpha 1-antitrypsin) phenotypes by a large-scale immobilized pH gradient. American J. Human Genetics. 37:922-930
Gu, M., and Du, X. (1998) A novel ligand-binding site in the ζ-form 14-3-3 protein recognizing the platelet glycoprotein Ibα and distinct from the c-Raf-binding site. J. Biol. Chem.273:33465-33471
Hansen, S.N., Waterfield, M.D., and Cramer, R. (2001) Proteomics-post- genomic cartography to understand gene function. TRENDS Pharmacol. Sci. 22:376-384
Jones, S.L.,and Brown, E.J. (1996) FCγRⅡ-mediated adhesion and phagocytosis induced L-plastin phosphorylation in human neutrophils. J. Biol. Chem. 271:14623-14630
Jones, S.L., Wang, J., Turck, C.W. and Brown, E.J. (1998) A role for the actin-binding protein L-plastin in the regulation of leukocyte integrin function. Proc. Natl. Acad. Sci. USA 95:9331-9336
Kanehisa, M., and Bork, P. (2003) Bioinformatics in the post-sequence era. Nat Genet. 33:305-10.
Keenan. C., and Kelleher, D. (1998) Protein kinase C and the cytoskeleton. Cell Signal. 10:225-32
Klose, J. (1975) Protein mapping by combined isoelectric fousing and electrophoresis of mouse tissue, a novel approach to testing for induced point mutation in mammals. Humangenetik 26:231-243
Klose, J. (1999) Large-scale 2-D electrophoresis. Methods Mol. Biol. 112:147-172
Lauber, W.M., Carroll, J.A., Dufield, D.R., Kisesl, J.R., Radabaugh, M.R., and Malone J.P. Mass spectrometry compatibility of two-dimensional gel protein stains. Electrophoresis 22:906-918
Levchenko, A. (2001) Computational cell biology in the post-genomic era. Molecular Biology Reports 28:83-89
Lopez, M.F., Berggren, K., Chernokalskaya, E., Lazarev, A., Robinson, M., and Patton, W.F. (2000) A comparison of silver stain and SYPRO Ruby Protein Gel Stain with respect to protein detection in two-dimensional gels and identification by peptide mass profiling. Electrophoresis. 21:3673-3683.
Munday, A.D., Berndt, M.C., and Mitchell, C.A. (2000) Phosphoinositide 3-kinase form a complex with platelet membrane glycoprotein Ib-IX-V complex and 14-3-3ζ . Blood 96:577-584
O’Farrel, F.H. (1975) Higt resolution two-dimensional electrophoresis of protein. J. Biol. Chem.250:4007
Paclet, M.H., Davis, C., Kotsonis, P., and Godovac-Zimmermann (2004) N-formylpeptide receptor subtypes in human neutrophils activate L-plastin phosphorylation through different signal transduction intermediates. Biochem. J. 377:469-477
Quadroni, M. and James, P. (1999) Proteomics and automation. Electrophoresis 20:664-677
Piubelli, C., Galvani, M., Hamdan, M., Domenici, E., and Righetti, P.G. (2002) Proteome analysis of rat polymorphonuclear leukocytes : A two-dimensional electrophorsis/mass spectrometry approach. Electrophoresis 23:298-310
Rabilloud, T., Adessi, C., Giraudel, A., and Lunardi, J. (1997) Improvement of the solubilization of proteins in two-dimensional electrophoresis with immobilized pH gradients. Electrophoresis 18:307-316
Rabilloud., T. (1998) Use of thiourea to increase the solubility of membrane proteins in two-dimensional electrophoresis. Electrophoresis 19:758-760
Ross, R. (1993) The pathogenesis of atherosclerosis: a perspective for the 1990s. Nature 362:801-809
Shan, H.D., and Goyal, R.K. (2004) Glycoprotein Ⅱb/Ⅲa receptor and its inhibition:A platelet-directed therapeutic strategy. Indian J. Pharmacol. 36:133-139
Shevchenko, A., Wilm,M., Vorm, O., and Mann, M. (1996) Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. Anal.Chem. 68:850-858
Steinberg, T.H., Haugland, R.P., and Singer, V.L. (1996) Applications of SYPRO orange and SYPRO red protein gel stains. Anal Biochem. 239:238-45.
Surks, H.K., Mochizuki, N., Kasai, Y., Georgescu, S.P., Tang, K.M., Ito,M., Lincoln, T.M., and Mendelsohn, M.E. (1999) Regulation of myosin phosphatase by a specific interaction with cGMP-dependent protein kinase Iα. Science 286:1583-1587
Wilkins, M. R., Sanchez, J. C.,Dooley, A. A.,Appel, P. D., Humphery-Smith, I., Hochstrasser, D. F. and Williams, K. L. (1996) Progress with proteome projecys: Why all proteins expressed by a genome should be identified and how to do it. Biotech. Genet. Eng. Rev. 13:19-50
Yan, J.X., Wait, R., Berkelman, T., Harry, R.A., Westbrook, J.A., Wheeler, C.H., and Dunn, M.J.(2000) A modified silver staining protocol for visualization of proteins compatible with matrix-assisted laser desorption/ionization and electrospray ionization-mass spectrometry. Electrophoresis 21:3666-3372
Yan, J.X., Harry, R.A., Spibey, C., and Dunn, M.J. (2000) Postelectrophoretic staining of proteins separated by two-dimensional gel electrophoresis using SYPRO dyes. Electrophoresis 21:3657-65.