建立測試抗黑色素過度沉著藥物的三維皮膚模擬器

Establishment of a 3D human skin equivalent for testing effect of anti-hyperpigmentation agents

周怡廷(Yi-Ting Chou)

指導教授 ： 何美泠

共同指導教授 ： 王惠民(Hui-Min Wang)

高雄醫學大學/醫學院/生理及分子醫學研究所/碩士(2010年)

摘要

篩選抗黑色素過度沉著藥劑平台，包括：蘑菇的酪胺酸酶及單層細胞模式。然而這些篩選模式無法反映出人體皮膚正常生理情形。此外，由於動物保護組織的提倡，歐盟自2009年3月起禁止以動物實驗來測試化粧品類研究。目前還未有生物工程製作的人工皮膚能確實模擬正常未受傷皮膚的生理情形。首先發現從白玉蘭純化出的天然物(-)-N-formylanonaine對於蘑菇的酪胺酸酶具有抑制效果。再以人類上皮黑色素細胞測試，發現亦能抑制其酪胺酸酶活性及黑色素生成，且對於細胞沒有顯著毒性。比其他已知的酪胺酸酶抑制劑，如麴酸(kojic acid)及苯基硫脲(1-phenyl-2-thiourea, PTU)更佳。我們並以玻尿酸、膠原蛋白與明膠製備了適合將角質細胞、黑色素細胞及皮膚纖維母細胞共同培養於其上的三維支架，分析了含水率及孔徑大小。三種細胞共同培養部分，以染色確定此皮膚模擬器之切片形態，建立篩選抗黑色素試劑的平台。天然物(-)-N-formylanonaine確實具抗黑色素過度沉著的效果，將繼續於我們新建立的三維皮膚模擬器上作進一步測試。

關鍵字

皮膚 ； 黑色素 ； 酪胺酸酶支架 ； 組織工程

並列摘要

Many methodologies such as enzymatic (mushroom tyrosinase) and monolayer cell culture assays are routinely used to screen novel de-pigmentary agents. These models have disadvantages in terms of physiological and economic relevance. Besides, due to the promotion of animal protection organizations, cosmetic experiments on animals had been forbidden by the European Union since March, 2009. At the present time, there are no models of bioengineered skin that completely replicate the physiological conditions of uninjured skin. A natural product, (-)-N-formylanonaine, was isolated from the leaves of Michelia alba D.C. (Magnolianceae). It was found to inhibit mushroom tyrosinase and was further tested on human epidermal melanocytes (MCs) to be with tyrosinase and melanin reducing activities in human epidermal MCs without apparent cytotoxicity to human cells, superior to the known tyrosinase inhibitors, such as kojic acid and 1-phenyl-2-thiourea (PTU). We fabricated an appropriate scaffold with hyaluronic acid, collagen, and gelatin for three kinds of cells, keratinocytes (KCs), MCs and fibroblasts (FBs) to co-culture on and tested the pore size and swelling ratios of this scaffold. As co-culturing three kinds of cells, the morphology was checked by paraffin section and staining to manufacture the de-pigmentation platform. The de-pigmentation effect of (-)-N-formylanonaine will be tested on our novel established platform of 3D human skin equivalent.

並列關鍵字

skin ； melanin ； tyrosinase ； scaffold ； tissue engineering

參考文獻

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