|a英文題名:Establish a Novel Depigmenting Compound Screening System Using Fluorescent Protein Reporter Combined with the Promoters of Microphthalmia-Associated Transcription Factor and Tyrosinase Gene Family
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|a指導教授:林智健
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|a含參考書目
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|a碩士論文--靜宜大學化粧品科學學系碩士班
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|a黑色素為保護人類皮膚不被陽光中的紫外線傷害之重要角色，其生合成主要受到酪胺酸酵素(Tyrosinase)、酪胺酸酵素相關蛋白-1 (Tyrosinase-related protein-1, TRP1)和酪胺酸酵素相關蛋白-2 (Tyrosinase-related protein-2, TRP2)等酪胺酸酵素家族之催化。此外，小眼症相關轉錄因子(Microphthalmia-associated transcription factor, MITF)調控這些酪胺酸酵素基因家族之轉錄。然而，過多的黑色素生成造成一些美觀上的問題，如雀斑、老人斑和肝斑，因此越來越多人渴望找到一些新型的美白成份。目前常見的篩選美白化合物之方法，如體外蘑菇酪胺酸酶活性分析和胞內黑色素含量分析，具有可信度與耗費多時之問題。因此本實驗希望建立一個可以改善上述問題的篩選系統。首先將增強型綠色螢光報導系統與小眼症相關轉錄因子、酪胺酸酵素家族基因之啟動子結合，經由轉染、篩選穩定細胞，加入熊果素(Arbutin)、鼠尾草酸(Carnosic acid)、α-促黑激素(α-MSH)、茶鹼(Theophylline)……等八種影響黑色素生成檢品，再藉由螢光讀值強弱分析此報導系統，同時黑色素含量測定進行比較。由結果顯示：(1) 熊果素、鼠尾草酸及α-促黑激素對於所有細胞螢光蛋白報導表現皆無影響，茶鹼隨濃度增加，MeWo-pMITF細胞之螢光表現量呈現劑量依存性增加；MeWo-pTyr細胞之螢光表現量些微上升；MeWo-pTRP2細胞之螢光表現量皆一致增高。(2) 熊果素與鼠尾草酸對B16-F10細胞黑色素含量隨劑量增加具劑量依存性降低，α-促黑激素對黑色素含量為增加，茶鹼對黑色素含量隨劑量上升具劑量依存性增加。以上結果與先前的研究相符，因此本研究建立之螢光蛋白報導系統具有可以快速地分析出化合物對黑色素生成之調控，且未來有機會應用於美白成份之篩選。|uMelanin plays an important role in protecting human skin against ultraviolet sun radiation damage, which was synthesized from tyrosine by the regulation of tyrosinase gene family, including tyrosinase, tyrosinase-related protein-1 (TRP1), and tyrosinase-related protein-2 (TRP2). Besides, microphthalmia-associated transcription factor (MITF) regulates the transcription of tyrosinase gene family. However, more melanin makes an esthetic problem, such as freckles, senile lentigines and melasma. More and more people concentrate on founding the novel skin-whitening agents. Screening methods for depigmenting compound, such as in vitro mushroom tyrosinase and cell-based melanin content assay, are presenting less satisfactory results and time-consuming procedures. To improve the disadvantages of these screening methods, we try to construct a novel screening method by the enhanced green fluorescent protein (EGFP) reporter system, combining with the promoters of MITF and tyrosinase gene family. We first confirmed the validity of this reporter assay system by using Arbutin, Carnosic acid, α-MSH, Theophylline…etc. The fluorescent intensities of MeWo-pMITF, MeWo-pTyr and MeWo-pTRP2 cells treated with Arbutin, Carnosic acid and α-MSH have no significant different from control. The fluorescent intensities of theophylline treated MeWo-pMITF, MeWo-pTyr and MeWo-pTRP2 cells were increased in a dose-dependent manner, increased slightly and increased significantly, respectively. Arbutin and Carnosic acid decreased melanogenesis, α-MSH and Theophylline strongly increased melanogenesis. These results indicate this novel screening system can be used as a convenient tool to discover the new depigmenting compounds and to investigate the hypopigmentation mechanism of the well-known skin-whitening agents.
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|aEstablish a Novel Depigmenting Compound Screening System Using Fluorescent Protein Reporter Combined with the Promoters of Microphthalmia-Associated Transcription Factor and Tyrosinase Gene Family
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