臺灣博碩士論文加值系統

生長因子做為醫藥用跟化妝品之用在生物科技方面是有趣的且是應用上其中的一部分。表皮生長因子(EGF)是生長因子類中的其中一種，而且對於在ectodermal和mesodermal層的不同種類細胞有著增生分裂的效果。EGF和FGF最近已經被使用為化妝品內的特定成分之一，然而，生長因子穩定性不佳的情況被視為限制醫藥和化妝品之用，且在合成、管理及保存上產生問題。因此加入穩定因子在EGF溶液內，藉由旋光光譜儀檢測熱處理過的人類生長因子構型以及使用流式細胞儀獲得細胞周期的分析來鑑定熱處理過的人類生長因子生物活性，由這兩種方式來評估哪個穩定因子能真正穩定EGF。而mannitol，polyacrylic acid (PAA)，heparin，fullerene-OH， polyvinylalcohol (PVA)，hydroxypropyl-cellulose (HPMC)和polymethacrylic acid (PMAA)這些物質對於EGF而言被選為最適合穩定因子的候選人。這個研究的目的是去發展hEGF穩定性的物理評估方法，且找到物理評估方法和hEGF生物活性檢測的關係性。hEGF的無摺疊比率由旋光光譜儀檢測，在232 nm下被定義出，而熱處理過的hEGF無摺疊比率不管在有沒有穩定因子的存在下，都會隨著溫度增加而增加。在無摺疊比率達80%下此時的溫度來判定哪個是真正穩定因子來穩定EGF的結構，我們得知heparin, PVA和mannitol是好的單一成分穩定因子來穩定EGF。再者，藉由流式細胞儀來鑑定hEGF的生物活性，我們成功找出hEGF物理性評估和生物活性的關係，而且發現mannitol，heparin和HPMC是好的穩定因子來穩定hEGF。總結hEGF的物理性評估和生物活性測定，mannitol，heparin和HPMC是合適的穩定因子來穩定hEGF。在化妝品實質應用上，十位試驗者在使用完四種不同化妝品之後，我們使用MPA580皮膚測試儀測定試驗者皮膚的彈性、黑色素含量、紅色素含量和油脂分泌量，而含有hEGF成份的化妝品能改善皮膚彈性，降低紅色素含量以及控制油脂分泌量。

Growth factors used as pharmaceuticals and cosmetics are one of the interesting and major applications in biotechnology. Epidermal growth factor, EGF, is one of growth factors, and is a potent mitogenic factor for a variety of cultured cells originated from ectodermal and mesodermal layers. EGF and FGF have been utilized as one of specific components in cosmetics recently. However, low stability of growth factors is considered to limit pharmaceutical and cosmetic applications of growth factors, generating problems in formation, administration and storage. Therefore, the effect of stabilizers in EGF solution was investigated on the stability of human EGF (hEGF) from conformation of heat-treated hEGF using circular dichroism (CD) spectroscopy and from bioactivity of heat-treated hEGF from cell cycle analysis using flow cytometry. The following stabilizers were selected as the candidates of optimal stabilizers for hEGF: mannitol, polyacrylic acid (PAA), heparin, polyvinylalcohol (PVA), fullerene-OH, hydroxypropyl-cellulose (HPMC) and polymethacrylic acid (PMAA). The goal of this research is to develop physical evaluation method of hEGF stability and activity, and to find the relationship between physical evaluation method and bioassay of hEGF activity. The unfolding fraction of hEGF measured by CD spectroscopy at 232nm increased with increasing heat-treated temperature of hEGF with and without stabilizers. The temperature of hEGF solution, which showed the temperature where the unfolding fraction of hEGF was 80% was also analyzed in hEGF solution containing no or 0.05 wt% of stabilizers, and shifted to higher temperature when hEGF solution contained heparin, PVA or mannitol. These results indicates heparin, PVA and mannitol are one of good candidates of hEGF stabilizers as single component stabilizers. PMAA, heparin and PVA are found to be good candidates of hEGF containing multi-component stabilizers to stabilize the conformation of PARS-hEGF (hEGF containing 0.31 wt% mannitol) compared to PARS-hEGF containing no or other stabilizers from unfolding fraction analysis of PARS-hEGF.
DNA synthesis of fibroblast cells was also analyzed from cell cycle measurements using flow cytometry, and was used as the bioassay of hEGF activity. We demonstrated physical evaluation method of hEGF stability and activity, and found the relationship between physical evaluation method and bioassay of hEGF activity. In the summary of combination between physical evaluation and bioassay for hEGF, mannitol, heparin and HPMC were found to be optimal stabilizers to stabilize hEGF.
The effect of cosmetics containing hEGF on human skin conditions was also evaluated from monitoring test for ten volunteers using cosmetics containing with and without hEGF as one of the application of hEGF in bioengineering. The elasticity, melanin amount, erythem amount and surface sebum amount of skin on the human volunteers were measured from cutometer MPA580 instrument. Cosmetic A containing hEGF was found to be effective to improve the elasticity, erythem amount and control of sebum amount on the human skin in this study.

Chapter 1 Introduction………..................................................................1
1-1 Research Background…………………………………………………………...2
1-1-1 Epidermal Growth Factor (EGF)………………………………………….2
1-1-2 EGF receptor………………………………………………………………..7
1-1-3 EGF Pathway………………………………………………………………10
1-1-4 Mitogenic-activated protein kinases (MAPKs) signaling cascades…….16
1-1-5 Cell cycle…………………………………………………………………...17
1-1-6 Circular Dichroism………………………………………………………..20
1-1-6-1 Principle of Circular Dichroism (CD)……………………………….20
1-1-6-2 Applications of CD spectroscopy…………………………………….22
1-1-7 Flow Cytometry……………………………………………………………25
Chapter 2 Materials & Methods………………………………………..28
2-1 Materials………………………………………………………………………..28
2-1-1 Chemicals………………………………………………………………….28
2-1-2 Cell line…………………………………………………………………….29
2-1-3 Consumables………………………………………………………………29
2-1-4 Instruments………………………………………………………………..30
2-2 Experimental Methods…………………………………………………………31
2-2-1 Preparation of PBS (phosphate buffer saline solution)…………………31
2-2-2 Preparation of PB (phosphate buffer)……………………………………31
2-2-3 Preparation of culture medium……………………………………..........32
2-2-4 Cell culture………………………………………………………………....32
2-2-5 Cell freezing………………………………………………………………..34
2-2-6 Cell thawing……………………………………………………………….34
2-2-7 Cell growth curve measurements………………………………………...34
2-2-8 Circular dichroism (CD) measurements…………………………………35
2-2-9 Cell treatment……………………………………………………………...35
2-2-10 Cell cycle measurements by flow cytometry……………………………36
2-2-11 Skin evaluation measurements…………………………………………..36
Chapter 3 Results & Discussion………………………………………...38
3-1 Cell growth curve of fibroblasts in several culture medium………………...38
3-1-1 Cell growth of mouse NIH Swiss embryo, (NIH/3T3) cells……………..38
3-1-2 Cell growth of human foreskin fibroblast (Hs68) cells………………….42
3-2 hEGF structure from Physical measurements……………………………….46
3-2-1 CD spactra and unfolding fractions of hEGF…………………………...46
3-2-2 CD spactra and unfolding fractions of hEGF with stabilizers………….49
3-2-2-1 CD spactra of hEGF with single stabilizer………………………….49
3-2-2-2 Relationship between unfolding fraction and secondary structure of
hEGF to investigate optimal stabilizers to stabilize hEGF…………58
3-2-3 CD spactra and unfolding fractions of hEGF containing 0.31 wt%
mannitol (PARS-hEGF) plus stabilizers…………………………………62
3-2-3-1 Physical characteristics of PARS-hEGF plus single stabilizer…….62
3-2-3-2 Relationship between unfolded fraction and secondary structure
analysis for adequate stabilizers to stabilize PARS………………..69
3-3 Bioactivity of hEGF………………………………………………….................73
3-3-1 Relationship between unfolding fraction and bioactivity for heat-treated
hEGF……………………………………………………………………….78
3-3-2 Relationship between unfolding fraction and bioactivity of heat-treated
hEGF at 80 oC with single stabilizers…………………………..………...81
3-4 Evaluation of effect of cosmetics including growth factors on improvement of
skin conditions……………………………………………………….………….85
3-4-1 Evaluation of elasticity on human skin………………………..…………89
3-4-2 Evaluation of melanin amount on human skin……………………..…...91
3-4-3 Evaluation of skins for the content of erythem…………………..……...93
3-4-4 Evaluation of skins for the content of sebum…………………..………..95
Chapter 4 Conclusion……………………………………………………97
Bibliography…………………………………………………………..100

[1] Graham Carpenter and Stanley Cohen. “Epidermal growth factor”, The Journal of Biological Chemistry 265 (14): 7709–7712, May 1990.
[2] Elizabeth S. Henson and Spencer B. Gibson. “Surviving cell death through epidermal growth factor (EGF) signal transduction pathways: Implications for cancer therapy”, Cellular Signalling 18 (2006) 2089–2097.
[3] M. Jost, C. Kari and U. Rodeck. “The EGF receptor – an essential regulator of multiple epidermal functions”, Eur. J. Dermatol. 10 (7) (2000) 505-510.
[4] A.W. Burgess. et al. “An open-and-shut case? Recent insights into the activation of EGF/ErbB receptors”, Mol. Cell 12 (3) (2003) 541-552.
[5] R.S. Herbst. “Review of epidermal growth factor receptor biology”, Int. J. Radiat. Oncol. Biol. Phys. 59 (2 Suppl) (2004) 21.
[6] Stanley Cohen. “Isolation of a mouse submaxillary gland protein accelerating incisor eruption and eyelid opening in the new-born animal”, J. Biol. Chem. 237, 1555-1562, 1962.
[7] John M. Taylor, William M. Mitchell, and Stanley Cohen. “Epidermal Growth Factor Physical and Chemical Properties”, The Journal of Biological Chemistry, Vol. 247, 5928-5934, 1972.
[8] Stanley Cohen and Graham Carpenter. ”Human Epidermal Growth factor: Isolation and Chemical and Biological Properties”, Proc. Natl. Acad. Sci. U.S.A. 72, 1317-1321, April 1975.
[9] H. Gregory. “Isolation and structure of urogastrone and its relationship to epidermal growth factor”, Nature, 257, 325-327, September 1975.

[10] Graham Carpenter and Stanley Cohen. “Human Epidermal Growth Factor and the Proliferation of Human Fibroblasts”, J. Cell. PHYSIOL. 88, 227-238, 1975.
[11] Leslie A. Holladay. et al. “Conformation and Unfolding Thermodynamics of Epidermal Growth Factor and Derivatives”, Biochemistry, Vol. 15, 2624-2633, 1976.
[12] Barnham KJ. et al. “Role of the 6-20 disulfide bridge in the structure and activity of epidermal growth factor”, Protein Sci. 7: 1738-1749, 1998.
[13] Dianne Alewood. et al. “The role of disulfide bonds in the structure and function of murine epidermal growth factor (mEGF)”, Growth Factors, 23(2), 97-110, June 2005.
[14] C. Richard Savage. et al. “Epidermal Growth Factor Location of Disulfide Bonds”, The Journal of Biological Chemistry, Vol. 248, No. 22, Issue of November 25, 7669-7672, 1973.
[15] Marlon R. Schneider and Eckhard Wolf. “The Epidermal Growth Factor Receptor Ligands at a Glance”, J. Cell. Physiol. 218: 460-466, 2009.
[16] Graham Carpenter and Stanley Cohen. “Epidermal Growth Factor”, Ann. Rev. Biochem. 48:193-216, 1979.
[17] Keiji Furuuchi. et al. “Targeted Antireceptor Therapy with Monoclonal Antibodies Leade to the Formation of Inactivated Tetrameric Forms of ErbB Receptors”, The Journal of Immunology, 178: 1021-1029, 2007.
[18] Schechter A. L. et al. “The neu oncogene: an erb-B-related gene encoding a 185,000-Mr tumour antigen”, Nature 312: 513–516, 1984.
[19] Kokai Y. et al. “Synergistic interaction of p185c-neu and the EGF receptor leads to transformation of rodent fibroblasts”, Cell 58: 287–292, 1989.

[20] Greene M. I. et al. “Receptor systems in tissues of the nervous system”, Immunol. Rev. 100:153–184, 1987.
[21] Kraus M. H. et al. “Isolation and characterization of ERBB3, a third member of the ERBB/epidermal growth factor receptor family: evidence for overexpression in a subset of human mammary tumors”, Proc. Natl. Acad. Sci. USA 86: 9193–9197, 1989.
[22] Yarden Y. and M. X. Sliwkowski. “Untangling the ErbB signalling network”,
Nat. Rev. Mol. Cell Biol. 2: 127–137, 2001.
[23] Thomas Grewal and Carlos Enrich. “Annexins – Modulators of EGF receptor signaling and trafficking”, Celluar Signalling, 21, 847-858, 2009.
[24] Alan Wells. “EGF receptor”, The International Journal of Biochemistry & Cell Biology, 31, 637-643, 1999.
[25] Kuhnen C and Winter BU. “EGFR-expression in pulmonary neuroendocrine cell hyperplasia”, Pathologe. 27(2):147-151, 2006 March.
[26] Smith KD, Wells A, Lauffenburger DA. “Multiple signaling pathways mediate compaction of collagen matrices by EGF-stimulated fibroblasts”, Exp Cell Res. 2006 July 1;312(11):1970-1982.
[27] Immervoll H. et al. “Molecular analysis of the EGFR-RAS-RAF pathway in pancreatic ductal adenocarcinomas: lack of mutations in the BRAF and EGFR genes.”, Virchows Arch. 2006 June;448(6):788-796.
[28] Meng S. et al. “Participation of both Gab1 and Gab2 in the activation of the ERK/MAPK pathway by epidermal growth factor”, Biochem J. 2005 Oct 1; 391(Pt 1):143-151.
[29] Wang Y, Wu J, Wang Z. “Akt binds to and phosphorylates phospholipase C-gamma1 in response to epidermal growth factor”, Mol Biol Cell. 2006 May;17(5):2267-2277.
[30] Alvarez JV. et al. “Signal transducer and activator of transcription 3 is required for the oncogenic effects of non-small-cell lung cancer-associated mutations of the epidermal growth factor receptor”, Cancer Res. 2006 March 15; 66(6): 3162-3168.
[31] Lanzetti L. et al. “The Eps8 protein coordinates EGF receptor signalling through Rac and trafficking through Rab5”, Nature. 2000 Nov 16;408(6810):374-377.
[32] Haglund K. et al. “Sprouty2 acts at the Cbl/CIN85 interface to inhibit epidermal growth factor receptor downregulation”, EMBO Rep. 2005 July;6(7):635-641.
[33] Beeser A. et al. “Role of group A p21-activated kinases in activation of extracellular-regulated kinase by growth factors”, J Biol Chem. 2005 Nov 4;280(44):36609-36615.
[34] Li Y. et al. “The epidermal growth factor receptor regulates interaction of the human DF3/MUC1 carcinoma antigen with c-Src and beta-catenin”, J Biol Chem. 2001 Sep 21;276(38):35239-35242.
[35] Ettinger DS. “Clinical implications of EGFR expression in the development and progression of solid tumors: focus on non-small cell lung cancer”, Oncologist. 2006 Apr;11(4):358-373.
[36] Paul M. Harari. et al. “Biology of Interactions: Antiepidermal Growth Factor Receptor Agents”, Journal of Clinical Oncology, Vol. 25, 4057-4065, September 10, 2007.
[37] Lufen Chang and Michael Karin. “Mammalian MAP kinase signaling cascades”, Nature, Vol. 410, 37-40, 1 March 2001.
[38] Bruce Alberts. et al. Molecular Biology of The Cell, Published by Garland Science, Taylor & Francis Group, Fifth Edition.
[39] Koji Nakanishi, Nina Berova and Robert W. Woody. Circular Dichroism Principles and Applications, VCH Publishers, Inc. 1994.
[40] V. P. Saxena and D. B. Wetlaufer. “A New Basis for Interpreting the Circular Dichroic Spectra of Proteins”, Proc Natl Acad Sci U S A. 1971 May; 68(5): 969–972
[41] Yang JT, Wu CS, Martinez HM. “Calculation of protein conformation from circular dichroism”, Methods Enzymol. 1986;130:208-269.
[42] Nathan P. Cowieson. et al. “Evaluating protein:protein complex formation using synchrotron radiation circular dichroism spectroscopy”, Proteins. 2008 Mar;70(4):1142-1146.
[43] S. Shankara Narayanan and Samir Kumar Pal. “Nonspecific Protein-DNA Interactions: Complexation of r-Chymotrypsin with a Genomic DNA”, Langmuir 2007, 23, 6712-6718.
[44] Ghosh KS. et al. “Sectroscopic investigation into the interactions of 3''-O-carboxy esters of thymidine with bovine serum albumin”, Biopolymers. 2009 Apr 28; 91(9): 737-744.
[45] Juskowiak B, Gałezowska E, Takenaka S. “Spectral properties and binding study of DNA complexes with a rigid bisintercalator 1,4-bis((N-methylquinolinium-4-yl)vinyl)benzene”, Spectrochim Acta A Mol Biomol Spectrosc. 2003 Mar 15;59(5):1083-1094.
[46] Milanesi L. et al. “A method for the reversible trapping of proteins in non-native
conformations”, Biochemistry. 2008 Dec 23;47(51):13620-13634.
[47] Munier-Lehmann H. et al. “Thymidylate kinase of Mycobacterium tuberculosis: a chimera sharing properties common to eukaryotic and bacterial enzymes.”, Protein Sci. 2001 Jun;10(6):1195-1205.
[48] Andrew D. Robertson and Kenneth P. Murphy. “Protein Structure and the Energetic of Protein Stability”, Chem. Rev. Vol. 97, 1251-1267, 1997.
[49] Flow Cytometry: A Practical Approach, 3rd Edition. (Practical Approach Series).
Edited by M.G. Ormerod. Oxford University Press (2000).
[50] Introduction to Flow Cytometry, First Paperback Edition. James V.Watson. Cambridge University Press. (2004).
[51] Malgorzata Zakrzewska. et al. “Design of fully active FGF-1 variants with increased stability”, Protein Engineering, Design & Selection, Vol. 17 no. 8, 603-611, 2004.
[52] Malgorzata Zakrzewska. et al. “Highly Stable Mutants of Human Fibroblast Growth Factor-1 Exhibit Prolonged Biological Action”, J. Mol. Biol. 352, 860-875, 2005.
[53] Rio Kita. et al. “Pinning of phase separation of aqueous solution of hydroxypropylmethylcellulose by gelation”, Physics Letters A 259 _1999. 302–307.
[54] J. H. Park. et al. “A potential role of connexin 43 in epidermal growth factor-induced proliferation of mouse embryonic stem cells: Involvement of Ca2+/PKC, p44/42 and p38 MAPKs pathways”, Cell Prolif. 2008, 41, 786–802.
[55] Zhi-Ming Han. et al. “Flow cytometric cell-cycle analysis of cultured fibroblasts from the giant panda, Ailuropoda melanoleuca L.”, Cell Biology International 27 (2003), 349–353.
[56] S.M. Teresa Hernandez-Sotomayor and Graham Carpenter. “Epidermal Growth Factor Receptor: Elements of Intracellular Communication”, J. Membrane Bio. Vol. 128, 81-89, 1992.

[57] Robert N. Jorissen. et al. “Epidermal growth factor receptor: mechanism of activation and signalling”, Experimental Cell Research, Vol.284, 31-53, 2003.
[58] Qun-sheng Ji. et al. “Epidermal Growth Factor Signaling and Mitogenesis in Plcg1 Null Mouse Embryonic Fibroblasts”, Molecular Biology of the Cell Vol. 9, 749–757, April 1997.
[59] Sang Jin Cheon. et al. “Effects of growth factors and kinase inhibitors on the properties of human adipose-stromal cells in different culture conditions”, Cell Biology International 32 (2008),784-791.
[60] Andrew D. Robertson and Kenneth P. Murphy. “Protein Structure and the Energetics of Protein Stability”, Chem. Rev. 1997, 97, 1251-1267.
[61] Ceren Alemdaroglu. et al. “Investigation of epidermal growth factor containing liposome formulation effects on burn wound healing”, Journal of biomedical materials research. Part A, vol. 85, 271-283, 2008.
[62] Gray Pearson. et al. “Mitogen-Activated Protein (MAP) Kinase Pathways: Regulation and Physiological Functions”, Endocrine Reviews 22(2): 153–183, April 2001.
[63] Gregory J. Wiepz. et al. “Growth Hormone Attenuation of Epidermal Growth Factor-Induced Mitogenesis”, JOURNAL OF CELLULAR PHYSIOLOGY, 173:44–53 (1997).
[64] Jui-Yoa Chang and Li Li. “The Disulfide Structure of Denatured Epidermal Growth Factor: Preparation of Scrambled Disulfide Isomers”, Journal of Protein Chemistry, Vol. 21, No. 3, March 2002.
[65] Gregory Parries. et al. “The Human Urinary Epidermal Growth Factor (EGF) Precursor”, THE JOURNAL OF BIOLOGICAL CHEMISTRY, Vol. 270, No. 46, Issue of November 17, 27954–27960, 1995.
[66] Stanley Cohen. “Origins of Growth Factors: NGF and EGF ”, THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. 283, NO. 49, 33793–33797, December 5, 2008.
[67] Caroline E. Gargett. et al. “Hormone and growth factor signaling in endometrial renewal: Role of stem/progenitor cells”, Molecular and Cellular Endocrinology 288 (2008) 22–29.
[68] Sun-Mi Kim. et al. “Effects of gangliosides on the differentiation of human mesenchymal stem cells into osteoblasts by modulating epidermal growth factor receptors”, Biochemical and Biophysical Research Communications 371 (2008) 866–871.
[69] Jeong Soon Lee. et al. “Heparin-binding epidermal growth factor-like growth factor inhibits adipocyte differentiation at commitment and early induction stages”, Differentiation (2008) 76:478–487.
[70] Akio Soeda. et al. “Epidermal Growth Factor Plays a Crucial Role in Mitogenic Regulation of Human Brain Tumor Stem Cells”, THE JOURNAL OF BIOLOGICAL CHEMISTRY, VOL. 283, NO. 16, 10958–10966, April 18, 2008.