- 學位論文
探討羥氯喹對皮膚燒燙傷治療及修復機制
The therapeutic mechanisms of Hydroxychloroquine on skin burn injurys
摘要
燒燙傷所引起的細胞死亡及傷口癒合的修復是近年來重要的研究議題。本研究欲探討內質網壓力及細胞自噬於燒燙傷中所扮演的角色，並分析羥氯喹對燒燙傷傷口的復原及影響。首先，我們透過西方墨點法分析燒燙傷後角質細胞所引起的內質網壓力及細胞自噬之相關蛋白表現，可發現燒燙傷後 BIP、BECLIN-1、P62、LC3B-Ⅰ之蛋白表現量下降，而 LC3B-Ⅱ則隨著燒燙傷時間增長而上升。同時透過流式細胞分析儀檢測，在燒燙傷溫度達到55℃以上，細胞會由凋亡轉為壞死，但在 48 小時後細胞的存活率上升，因此，我們透過西方墨點法分析細胞生長週期之相關蛋白，發現在48小時後 CDK6 蛋白有少量的上升，同時亦使用 Wnt/β-catenin 抑制劑 ICG001 觀察燒燙傷後傷口癒合的蛋白表現及細胞自噬與細胞移行之現象，透過實驗結果我們發現羥氯喹可以恢復燒燙傷後細胞之移行能力，而β-catenin 抑制劑及 mitomycin C 的共同處理，會使細胞加劇凋亡現象。最後，在燒燙傷模式的動物實驗中，含有羥氯喹的敷料，可有效促進大鼠傷口之癒合，並減少 CD11C 的表現及發炎現象。綜合以上結果，可得知在細胞實驗在燒燙傷中，羥氯喹在西方墨點法中無明顯的增殖作用表現，但在傷口癒合可促進細胞增殖及移行能力，而在動物實驗中，可鞋改善傷口發炎及促進癒合。
並列摘要
Repair of burn-induced injury on cell death and wound healing is an important research issue in recent years. We want to investigate the role of endoplasmic reticulum stress and autophagy in burn injury, and analyzing the recovery and effects of hydroxychloroquine on burns. In keratinocytes and fibroblast, LC3B-Ⅱwere increased in burn injury. BECLIN-1, P62, BIP, CDK6, p-Rb were decreased in burn injury. Thus, we analyzed of heat-induced cell including human keratinocytes and fibroblast on AnnexinⅤ/PI to detected cell apoptosis and necrosis via Flow Cytometer. When temperature raise to 55℃or higher than 55℃, cells went to necrosis. Furthermore, β-catenin inhibitor ICG001 and mitomycin C combined with hydroxychloroquine were used to observe cell migration of human keratinocytes in wound healing assay. The migration of heat-induced human keratinocyte was improved by hydroxychloroquine in wound healing assay. Combine with ICG001 and mitomycin C stimulating cell apoptosis. In animal model, Hydroxychloroquine promoted second-degree burns wound healing and reduce inflammation in rats. Hydroxychloroquine has no proliferative effect obviously in Western Blot, but promoting cell proliferation and migration ability in wound healing assay and animal experiments.