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    題名: 人類表皮成長因子的結構穩定性及生物活性測定;Stabilization of human Epidermal Growth Factor (hEGF) and Its Bioassay
    作者: 李仁豪;Jen-Hao Lee
    貢獻者: 化學工程與材料工程研究所
    關鍵詞: 表皮生長因子;EGF;Epidermal growth factor
    日期: 2009-06-25
    上傳時間: 2009-09-21 12:31:46 (UTC+8)
    出版者: 國立中央大學圖書館
    摘要: 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.
    顯示於類別:[化學工程與材料工程研究所] 博碩士論文

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