臺灣博碩士論文加值系統

接觸性皮膚炎主要分為兩種，一種為刺激性接觸性皮膚炎(ICD)，另一種為過敏性接觸性皮膚炎(ACD)。過去有文獻報導六價鉻是一個常見的過敏原，並且普遍使用於許多工業的生產過程。而本篇的研究目的在於暸解天然抗氧化物紫檀芪是否能有效地抑制六價鉻所誘發的過敏性接觸性皮膚炎進行機轉研究。在細胞試驗中，所使用的細胞株為 HaCaT 細胞 (正常人類角質層細胞)，實驗將給予六價鉻及紫檀芪處理，並進行後續分析。鈣離子外流是內質網壓力產生的指標之一，是以酵素免疫分析法偵測螢光強度。以 MMT assay 分析細胞存活率，並利用流式細胞儀分析細胞凋亡、自體吞噬以及細胞週期的分佈。另外，也利用西方墨點法分析內質網壓力、細胞凋亡、自體吞噬相關蛋白的表現量。動物試驗方面，使用C57BL/6母鼠，適應約兩週後再進行實驗。首先以管餵 (gavage feeding) 的方式每日給予紫檀芪持續五週，直至實驗結束。在第14天，於小鼠的兩處鼠蹊部外側施打六價鉻作為致敏測試 (Sensitization test) 。接著在第28天以皮下注射給予Boost dose。在第35天，以塗抹六價鉻的方式處理已敏化的小鼠作為誘發測試 (Elicitation test)。在誘發測試結束後的24、48及72小時，進行耳朵厚度的量測以及ACD反應的觀察和紀錄。第72小時觀察結束後犧牲動物，並且收集小鼠的耳朵，將此檢體的皮膚切片進行蘇木紫伊紅染色 (hematoxylin and eosin) 及組織免疫染色 (Immunohistochemistry)。首先，在偵測鈣離子的結果發現，低劑量暴露六價鉻的組別，鈣離子釋放的現象較為明顯。在細胞週期分佈的分析發現，經過六價鉻暴露24小時後，sub G0/G1期有增加的現象，而在細胞凋亡的分析上有明顯的劑量效應關係。給予紫檀芪處理後，可以顯著的減少細胞凋亡及細胞毒性，並促進自體吞噬的發生。而在西方墨點法結果顯示，紫檀芪可以降低 cleaved-caspase-3 以及 cleaved-PARP (細胞凋亡相關蛋白) 的表現量。此外，經過紫檀芪和自體吞噬抑制劑3-MA處理後，可以發現自體吞噬及細胞激素IL-1b之間的交互作用。在體內實驗中，本研究已建立六價鉻誘發過敏性接觸性皮膚炎的小鼠模式，並且發現給予紫檀芪處理後，可以有效抑制六價鉻誘發皮膚發炎的現象。綜合以上結果，紫檀芪可以有效的降低六價鉻所誘發的細胞凋亡、提高細胞存活率，並透過促進自體吞噬的方式抑制細胞激素IL-1b。因此紫檀芪對六價鉻所誘發的過敏性接觸性皮膚炎可能是一個有潛力的化學預防物質。

Contactl dermatitis could be divided into two types, one is irritant contact dermatitis (ICD), the other is allergic contact dermatitis (ACD). Previous studies have shown that hexavalent chromium (Cr6+) is a common allergen, and commonly used in numerous industrial processes. The purpose of this study is to investigate whether chromium-induced ACD could be inhibited effectively by treating nature antioxidant pterostilbene (PT). In in vitro study, HaCaT cells ( human normal keratinocyte) was treated with Cr6+ and PT. Ca2+ efflux was analyzed by fluoroskan ascent FL. Cell viability was examined by MTT assay. Apoptosis, autophagy and cell cycle distribution were analyzed by flow cytometry. Western blotting was used to determine ER stess-, apoptotic- and autophagic- associated proteins expression. In in vivo study, the female C57BL/6 mice were acclimated for 2 weeks before the experiments. C57BL/6 mice received PT and N-acetyl-cysteine (NAC) continued for five weeks by oral administration using syringe and needle for gavage feeding injection until the elicitation finished. On day 14, C57BL/6 mice were injected Cr6+ into each of the inguinal flanks as the sensitization then underwent boost and epicutaneous elicitation on day 28 and 35. ACD reactions were quantified by measuring the ear thickness at 24, 48 and 72 hours after the elicitation. Swelling and erthema were also recorded by photography and by hematoxylin and eosin (H＆E) and immunohistochemial (IHC) staining of skin sections of ears that had been collected at 72 hours after the elicitation test. Firstly, the results indicated HaCaT cells exposed to low dose of Cr6+ treatment for 4 hours increased Ca2+ efflux. The percentage of HaCaT cells in the sub G0/G1 phase were observed after Cr6+ treatment for 24 hours. We found a significant increase in apoptosis percentage in a dose-dependent manner, but not in autophagy. The Cr6+ treatment of HaCaT cells pretreated with PT resulted in a significant reduction in apoptotic cells and cytotoxicity. However there was a significant increase in autophagy. Furthermore, PT could decrease the expression of cleaved-caspase-3 and cleaved-PARP (apoptosis-related protein) which were activated by Cr6+. In addition, we demonstrated the interaction between autophagy and IL-1b after treated with 3-MA and PT. In in vivo study, epidermal Cr6+ administration causes cutaneous inflammation in mice skin. Nevertheless, PT significantly attenuated the Cr6+-induced skin inflammation in mice. The above observation indicated that HaCaT cells pretreated with PT could inhibit Cr6+-induced apoptosis, rescued cell survival and decreased IL-1b by promoting autophagy. PT could be a potential chemopreventive agent for the prevention of Cr6+-induced cytotoxicity and ACD.

第一章、序論1
第二章、文獻回顧2
第一節、六價鉻2
第二節、過敏性接觸性皮膚炎(ACD) 3
第三節、內質網壓力(Endoplasmic reticulum stress) 4
第四節、過敏性接觸性皮膚炎與細胞激素6
第五節、六價鉻所誘導的細胞自體吞噬與細胞激素7
第六節、抗氧化物與紫檀芪9
第七節、動物模式與HaCaT cells人類角質層細胞株11
第三章、研究目的12
第四章、研究架構13
第一節、In vitro 13
第二節、In vivo 15
第五章、研究材料與方法17
第一節、研究材料17
第二節、研究方法22
In vitro 22
(一)細胞培養22
(二)細胞解凍22
(三)細胞繼代培養22
(四)細胞冷凍22
(五)細胞計數23
(六)MTT assay 23
(七)偵測細胞內鈣離子23
(八)細胞週期分析24
(九)細胞凋亡分析24
(十)自體吞噬分析25
(十一)螢光顯微鏡分析自體吞噬現象25
(十二)西方墨點法25
(十三)RT-PCR 27
In vivo 30
第三節、統計分析33
第六章、實驗結果34
第一節、六價鉻對於人類角質層細胞之細胞毒性的劑量效應34
第二節、分析六價鉻對於人類角質層細胞所誘發之內質網壓力34
第三節、六價鉻對於人類角質層細胞所誘發MAPK細胞訊息傳遞路徑35
第四節、六價鉻對人類角質層細胞造成的細胞週期變化及與細胞凋亡(Apoptosis) 現象35
第五節、紫檀芪對六價鉻暴露之人類角質層細胞造成細胞毒性及細胞凋亡的影響36
第六節、六價鉻對於人類角質層細胞產生細胞自體吞噬 (Autophagy)現象37
第七節、紫檀芪對六價鉻暴露之人類角質層細胞造成自體吞噬現象之變化38
第八節、人類角質層細胞的自體吞噬現象與細胞激素的交互作用38
第九節、紫檀芪對於六價鉻所誘發過敏性接觸性皮膚炎的抑制作用39
第七章、討論40
第八章、結論43
第九章、參考文獻44
圖表50

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