探討Oxazolone引發小鼠異位性皮膚炎及對人類角質細胞株HaCaT之影響

Explore the Effects of Oxazolone Induced Mice Atopic Dermatitis and Human Keratinocyte HaCaT Cell Line

齊俐安(Li-Ann Chi)

指導教授 ： 張基隆

高雄醫學大學/醫學院/生物化學研究所/碩士(2014年)

摘要

異位性皮膚炎( Atopic dermatitis )是一種常見的皮膚免疫失調疾病，全球盛行率約10 - 20%，而在台灣盛行率約8 - 10 %，主要好發於嬰幼兒及青少年。有文獻指出，病徵起因與患者遺傳過敏性體質、外在環境過敏原及皮膚表皮層的屏障缺陷相關。而皮膚屏障缺陷，讓皮膚水分不斷流失，造成皮膚紅腫、乾燥及搔癢造成表皮受損，進而使環境中的過敏原容易侵入皮膚，導致皮膚產生過度的免疫反應造成過敏。到目前為止其詳細作用機轉尚未完全釐清。由文獻得知4-Ethoxymethylene-2-phenyl-2-oxazolin-5-one ( Oxazolone )能使皮膚快速產生免疫過敏反應，常用來做為誘發皮膚過敏反應模式的藥物。因此，本研究以Oxazolone塗抹在小鼠的耳朵來誘導免疫發炎症狀作為模擬異位性皮膚炎的模式，另一方面，也探討Oxazolone對人類正常角質細胞株( HaCaT )的影響。
　　在HaCaT細胞研究中，主要針對在Oxazolone作用下，對細胞的細胞毒性、氧化壓力、能量代謝、細胞凋亡、皮膚表皮基質蛋白Filaggrin及細胞因子IL-5等部分作分析探討。另外，BABL/c小鼠模式中，以0.8% ~ 1.6% Oxazolone塗抹小鼠耳朵4週，實驗期間，對其耳朵外觀、厚度及長度進行觀察和量測，分析耳朵組織的改變。
　　實驗結果顯示，人類角質細胞株HaCaT在Oxazolone濃度30、100、300、600及1,000 μM作用後，濃度高於300 μM細胞型態明顯改變、細胞存活率降低。在氧化壓力部分，在濃度高於300 μM時ROS表現增加，另外抗氧化物質榖胱甘肽( Glutathione )及調控轉錄因子Nrf-2表現降低。在能量代謝上，細胞在濃度高於600 μM ATP表現降低，相對調控糖解作用之相關蛋白p53及TIGAR表現也降低。而在細胞凋亡中，首先發現Oxazolone在濃度高於600 μM會引起細胞DNA片段化，另外Hoechst染色中，細胞凋亡小體數量增加。粒線體膜電位在濃度高於300 μM表現下降，細胞週期中Sub-G1在濃度高於300 μM表現增加，而Western blot實驗結果也顯示，Cytochrome c及促細胞凋蛋白Bax表現增加，Pro-caspase-9、Pro-caspase-3及抗凋亡蛋白Bcl-XL有表現下降的趨勢。皮膚表皮基質蛋白Filaggrin表現顯著下降及過敏反應相關細胞因子IL-5表現增加。而小鼠耳朵在Oxazolone作用刺激7天小鼠的耳朵有明顯泛紅、腫脹的現象，第15天小鼠的耳朵則開始有搔癢、皮膚脆弱，且小鼠耳朵外型也有逐漸萎縮的情形，表示耳朵組織中部分基質可能遭受破壞。
　　綜合上述實驗結果得知，在Oxazolone的刺激作用下，能使小鼠耳朵誘發免疫發炎反應其免疫表現相似於異位性皮膚炎。此外，在HaCaT細胞實驗結果也顯示，細胞開始產生凋亡的現象是由於受到氧化壓力的刺激，使細胞內能量代謝降低，造成粒線體膜電位下降。經由Caspapse-9、Caspase-3路徑調控皮膚表皮基質蛋白Filaggrin表現下降，細胞開始產生凋亡的情形，使抗氧化物質榖胱甘肽( Glutathione )表現量下降，並且誘導角質細胞產生過敏及發炎。

關鍵字

異位性皮膚炎 ； 角質細胞 ； Oxazolone

並列摘要

Atopic dermatitis is a common skin disease of immune disorder with prevalence of approximate 10 - 20% in the word and of approximate 8 - 10% in Taiwan, which mainly occurs in children and adolescents. Previous study indicated that its symptoms owe to constitution epidermis skin barrier defect to cause moisture loss resulting in dry, irritation, and itchy, that makes environmental allergens easily penetrating skin and inducing excessive allergic immune response. Up to now, the mechanisms of this action has not well been identified. Studies reported Oxazolone ( 4-Ethoxy methylene-2-phenyl-2-oxazolin-5-one ) may induce immune response leading to allergic skin, so it is suggested to be a stimulus for allergic reaction. The present study applied oxazolone on the mice ear to serve as an animal model of atopic dermatitis; in addition the effects of oxazolone on human keratinocyte cell line ( HaCaT ) were also explored, by which we hoped to investigate the mechanism of atopic dermatitis.
　　Results showed that, in HaCaT cells, oxazolone at concentration greater than 300 μM could significantly change cell’s morphology and reduce cell’s viability as well as increase ROS production and IL-5 expression. In addition, the mitochondria membrane potential, antioxidant glutathione contents, transcription factor Nrf-2 expression, and epidermal matrix protein filaggrin expression were also decreased. Furthermore, , apoptotic body and sub-G1 cell population in cell cycle were also increased suggesting apopotosis was induced, which was confirmed by Western blotting with the increased cytochrome c and Bax as well as the decreased Pro-caspase-9, Pro-caspase-3, and Bcl-XL expressions. At the concentra-tion higher than 600 μM, oxazolone could decrease ATP contents and glycolysis-related proteins expressions including p53 and TIGAR suggesting energy metabolism might be interfered in. In mice model, ear was reddish and swelling after oxazolone treatment for 7 days. After 15 days treatment, ear became shrinking suggesting matrix protein was damaged.
　　Herein we found oxazolone stimulation could interfere with energy metabolism, induce apoptosis and increase oxidative stress of keratinocyte cells. In respect of research, oxazolone stimulation either in HaCaT cell line or in mice ear may provide a model for study atopic dermatitis.

並列關鍵字

Atopic dermatitis ； Keratinocyte ； Oxazolone

參考文獻

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