- 學位論文
纖維母細胞與角質細胞燒燙傷治療及其修復機制之探討
The mechanisms of heat-induced cell death and recovery on skin burn wound healing in human Fibroblasts and Keratinocytes
摘要
修復燒燙傷所引起的細胞死亡以及促進傷口癒合是近年來重要的議題。首先，為釐清燒燙傷所引起細胞命運機制在皮膚細胞中所扮演的角色，以開發適當藥劑治療之。我們透過西方墨點法分析燒燙傷後皮膚細胞的細胞自噬、細胞凋亡與內質網壓力相關之蛋白表現量，發現燒燙傷後細胞中的LC3-II與Cleave-caspase-7表現量會上升且Beclin-1、p62及Grp78表現量下降。而在Atg5-/- MEF細胞中，發現若是缺乏細胞自噬能力會使得燒燙傷後的細胞凋亡現象更加嚴重。除此之外，透過西方墨點法分析，將重組人類Wnt3a蛋白使用於角質細胞可回復因燒燙傷所流失的β-catenin表現量，同時，在細胞癒合試驗中發現重組人類Wnt3a蛋白可回復角質細胞在燒燙傷後的移行能力，且上述作用皆可被β-catenin抑制劑ICG001所逆轉，而Wnt訊息路徑抑制劑IWP4則會加劇人類纖維母細胞在燒燙傷後所產生的細胞凋亡現象。另外，透過流式細胞儀分析，我們發現細胞自噬抑制劑氯奎寧與3-甲基腺嘌呤可減緩人類纖維母細胞於燒燙傷所引起的細胞凋亡，而在細胞癒合試驗中，發現氯奎寧也可改善角質細胞在燒燙傷後的移行能力，最後，在燒燙傷動物模式中，含有氯奎寧的敷料可以促進大鼠燒燙傷傷口癒合。綜合上述，可知在人類纖維母細胞與角質細胞在燒燙傷中，細胞自噬與Wnt/β-catenin訊息路徑在細胞修復與傷口癒合上扮演重要的角色。
並列摘要
Repair the burn-injury induced cell death and improve wound healing are the most important issue. To elucidate the role of cell death on heat-induced injuries in skin cells, we investigated the expression of autophagy, apoptosis and endoplasmic reticulum stress related proteins in skin cells on Western Blot. In fibroblasts, LC3-II and cleaced-caspase-7 were increased on burn injury. Beclin-1, p62 and Grp78 were decreased on burn injury. Blockage of heat-induced autophagy initiation increased apoptotic cell death in Atg5-/- mouse embryonic fibroblasts. In addition, recombinant human Wnt3a proteins ameliorated heat-reduced β-catenin loss on Western Blot and promoted migration of human keratinocytes in wound healing assay. β-catenin loss and wound healing could also be reversed by β-catenin inhibitor ICG001. Wnt pathway inhibitor, IWP4, increased heat-induced apoptosis in human fibroblasts on Annexin V/PI staining and flow cytometry analysis. In addition, autophagy inhibitors, Chloroquine and 3-Methyladenine, alleviated heat-induced apoptosis in human fibroblasts on Annexin V/PI staining and flow cytometry analysis. The migration of heat-injured human keratinocytes were improved by chloroquine in wound healing assay. In the burned animal model, chloroquine promoted second burn wound healing in rats. These findings suggest that regulation of autophagy and Wnt/β-catenin pathway play an important role in cell survival and wound healing on heat-induced injuries in human skin fibroblasts and human keratinocytes.