2026-11-092026-11-09https://scholars.csmu.edu.tw/handle/123456789/38639Alpha-hydroxy acids (AHAs) has been widely used in cosmetic agents and superficial chemical peeling for approximately 20 years. However, the biologic effects of AHAs on human keratinocytes and fibroblasts are limited. We have demonstrated that glycolic acid and lactic acid induced cell cycle arrest and apoptosis on human keratinocyte cell line (HaCaT)(in preparation for submission to publication, BJD and Dermatol Surg). Sheu et al. disclosed that WOX1 may play an important role in both physiological and pathological cell death of keratinocytes. Okano et al. found that GA accelerated collagen I synthesis in fibroblast, and modulates matrix degradation through keratinocyte-released cytokines. For a comprehensive investigation of the biologic effects of AHAs (glycolic acid, GA and lactic acid, LA) including: (1) The role of the WOX1 in the pathogenesis of GA/LA-induced apoptosis on HaCaT cells. (2) The effects of GA/LA on the expressions of MMPs and collagen I synthesis in various ages of human skin fibroblasts in vitro. (3) The effects of keratinocyte-derived cytokines on the expressions of MMPs and collagen I synthesis in human skin fibroblasts in vitro. (4) Biologic effects including apoptosis, cytokines secretion, expressions of MMPs and collagen I on GA/LA-treated, UVB-treated and GA/LA+UVB-treated SKH-hr1 mouse skin in vivo. The investigations will be carried out in 3 divided years: The 1st year: 1. We will use the human keratinocyte cell line (HaCaT), cultured human skin keratinocytes and fibroblasts to investigate the effects of GA/LA. We will use real time RT-PCR and Western blot to measure the expressions of the mRNA and protein levels of WOX1, and use confocal microscopy to detect the localization of WOX1, EndoG and cytochrome c to further elucidate the molecular pathways of GA/LA-induced apoptosis in keratinocytes. 2. Then, we will check the expressions of MMPs and collagen I via Western blot and real time RT-PCR in various ages of GA/LA-treated fibroblasts. 3. In addition, we will collect the supernatants of GA/LA-treated HaCaT and Keratinocytes to measure the levels of cytokines (TNF-α,IL-1α and IL-8) with enzyme immunoassay (EIA). The 2nd year: 1. To investigate the effects of GA/LA on the expressions of MMPs and collagen I in fibroblasts of various ages which is affected a direct effect or affected indirectly via keritinocyte-derived cytokines (TNF-α,IL-1α and IL-8). 2. To better understand the role of WOX1 in GA/LA treated HaCaT cells, We use siRNA technique to knockdown the WOX1 in HaCaT cells. We will collect WOX1-intact and WOX1-knockdown cells treated with GA/LA to investigate differentiate effects on apoptosis and its molecular pathways via different methods including flow cytometry, TUNEL assay, Western blot and real time RT-PCR. 3. We will investigate the expressions of cytokines (TNF-α,IL-1α and IL-8) in supernatants of cultured WOX1-knockdown HaCaT cells. Furthermore, we will use the supernatants onto cultured human skin fibroblasts treated or non-treated with GA/LA to measure the levels of changes of MMPs and collagen I. The 3rd year: 1. We will use hairless SKH-hr1 mouse as an in vivo model to investigate the biological effects and apoptotic effects of GA/LA and ultraviolet B (UVB) on mouse skin. The experiment will be performed in 3 divided groups: (1) UVB-treated (with various dosages of UVB irradiation) (2) topical GA/LA-treated (with different concentrations of GA/LA) (3) topical GA/LA+UVB-treated. We will take skin biopsy in time at different treatment protocols. 2. The removed skin in 3 divided groups will be processed for (1) Morphological examinations including stratum corneum, thickness of epidermis and dermis, and pathological changes. (2) Immunohistochemical stain for TNF-α, IL-1α and WOX1. (3) TUNEL assay to detect the occurrence of apoptosis (4) Western blotting to detect the expressions of MMPs, collagen I and WOX1 etc.果酸(α-hydroxy acids)被廣泛應用於化妝品與皮膚淺層換膚之臨床治療，但是果酸對皮 膚角質細胞及纖維母細胞的分子生物效應目前仍不十分清楚。本研究室過去的研究發現甘醇酸 與乳酸均可導致人類皮膚角質細胞 (HaCaT cell)的細胞週期停滯與凋亡作用，許漢銘等人的研 究顯示WOX1蛋白在表皮細胞之凋亡過程中也扮演重要角色。Okano 等人發現甘醇酸刺激角質 細胞cytokines分泌並調節纖維母細胞collagen I 及MMP的表現。為進一步系統性的探討果酸 (甘醇酸與乳酸)對皮膚角質細胞及纖維母細胞的分子生物效應，包括(1)果酸(GA/LA)引起角質 細胞的多重路徑細胞凋亡中，WOX1 扮演何種角色，(2)果酸(GA/LA)對不同年齡纖維母細胞 MMPs 及collagen I 的影響，(3)角質細胞分泌的細胞激素(cytokines)對纖維母細胞MMPs 及 collagen I 的影響，(4) 以無毛小鼠(Hairless SKH-hr1 )為研究果酸(GA/LA)與紫外線B(UVB) 對皮膚細胞的細胞凋亡及相關生物效應的動物模式。本研究擬分三年進行： 第一年： 1. 釐清果酸(GA/LA)引發人類皮膚角質細胞凋亡作用的分子機轉，並探討果酸對角質細胞 凋亡作用中WOX1的角色：以人類皮膚角質細胞株(HaCaT)及收集不同年齡層的正常人類 皮膚的角質細胞及纖維母細胞，加入不同濃度果酸(GA/LA)處理後，利用共軛顯微鏡技 術(confocal microscopy)分析WOX1、EndoG、cytochrome c 等蛋白在細胞中分布是否 發生改變，此外以real-time RT-PCR及Western blot分析WOX1 基因mRNA及蛋白表現 量，探討果酸對角質細胞凋亡作用中WOX1的角色。 2. 果酸作用於不同年齡層纖維母細胞MMPs 及膠原蛋白表現的影響：用Western blot 及 real time RT-PCR的方法測定果酸是否直接影響不同年齡層纖維母細胞MMPs及膠原蛋 白的表現。 3. 測定果酸(GA/LA)對角質細胞分泌細胞激素的影響：收集角質細胞HaCaT 細胞培養液 (supernatant)，以酵素免疫分析法(ELISA)比較果酸處理前後角質細胞HaCaT 培養液 中細胞激素(例如TNF-α,IL-1和IL-8等)含量的差異。 第二年： 1. 擬探討果酸(GA/LA)是否藉由增加HaCaT cell 細胞激素(例如TNF-α, IL-1α 和IL-8) 分泌而對不同年齡層纖維母細胞的基質代謝(MMPs及膠原蛋白)的影響。 2. 並進一步利用siRNA技術，抑制角質細胞(HaCaT cell) WOX1的表現，研究以果酸處理 經WOX1 knockdown的HaCaT cell，以流式細胞儀、TUNEL assay、Western blot及real time RT-PCR的方法，並比較其細胞凋亡的程度與路徑是否不同？有何不同？ 3. 進一步比較經WOX1 knockdown 的HaCaT cell 其細胞培養液(supernatant)對纖維母細 胞的基質代謝(MMPs及膠原蛋白)的影響是否有明顯差異？ 第三年： 以無毛小鼠(Hairless SKH-hr1)為研究果酸(GA/LA)與紫外線B(UVB)對皮膚細胞的細胞凋 亡及相關生物效應的動物模式。實驗將分成三組(1)照射UVB(不同UVB 劑量照射)(2)塗抹果酸 (不同濃度果酸)(3)塗抹果酸+照射UVB，在無毛鼠的皮膚分別進行觀察，在不同時間點、不同組 別之小鼠，取其皮膚，做下列之比較研究： (1)病理觀察：皮膚角質層、表皮層，及真皮層的厚度及變化(stratum corneum, thickness of epidermis and dermis, and pathological changes). (2)免疫組織化學染色(Immunohistochemical stain)：TNF-α, IL-1α and WOX1在皮膚的表現. (3)TUNEL assay：分析細胞是否發生凋亡(to detect the occurrence of apoptosis). (4)Western blotting偵測MMPs, collagen I and WOX1等蛋白在皮膚表現. 本計劃的執行希望能藉由對果酸(GA/LA)對培養的人類角質細胞與纖維母細胞的生物效應， 以及對於無毛鼠皮膚作用的研究，了解果酸對於皮膚細胞凋亡作用與角質細胞分泌的細胞激素對 纖維母細胞基質代謝的影響，有助於更加釐清果酸對人類皮膚細胞的生物效應。果酸甘醇酸乳酸HaCaT細胞皮膚角質細胞纖維母細胞細胞凋亡紫外線BSKH-hr1無毛鼠WOX-1TNF-αIL-1αAlpha-hydroxyacids (AHAs)Glycolic acid(GA)Lactic acid(LA)Ultraviolet B (UVB)HaCaT cellApoptosisHairless SKH-hr1 mouseWOX1TNF-αIL-1αProject