- 學位論文
探討秋水仙素對毛囊毛乳突細胞生長之影響
The Investigation of Effect of Colchicine on Hair Follicle Dermal Papilla Cells
摘要
藥物引起落髮是許多臨床用藥常見的副作用之ㄧ，不僅造成病患心理沮喪與降低其生活品質，且往往導致病患癒後不理想，目前這種副作用仍無法解決。已知秋水仙素在臨床上會造成病患落髮，但參與秋水仙素造成落髮的分泌因子與其作用機制，依然不很清楚。毛囊毛乳突細胞控制毛囊發育、毛髮生長與毛髮生長週期。本研究利用分泌質體學和蛋白質體學方法探討毛乳突細胞經秋水仙素作用後的條件化培養基，再用西方點墨法與即時定量聚合酶連鎖反應探討秋水仙素刺激毛乳突細胞分泌因子對毛乳突細胞的作用機制。本研究發現以2 μM 的秋水仙素處理毛乳突細胞六小時後得到的秋水仙素條件化培養基，可以抑制毛乳突細胞生長，而且秋水仙素與該秋水仙素條件化培養基皆會降低毛乳突細胞的鹼性磷酸酶活性。經分子篩離心管與液相層析儀純化後，發現有一個對熱且對胰蛋白酶敏感的蛋白質抑制因子。此蛋白質抑制因子經LC-ESI MS/MS 質譜儀分析與Mascot 資料庫比對鑑定後，再以西方點墨法分析，確定此蛋白質抑制因子為histone H4 (gi|4505821)；此外，本研究發現重組histone H4 也會降低毛乳突細胞的增生。此外，二維電泳分析顯示毛乳突細胞受到秋水仙素作用後，造成共24 個蛋白質表現量差異2 倍以上。本研究推測秋水仙素造成的落髮不僅與秋水仙素藥物本身有直接關係，並且與毛乳突細胞受到秋水仙素刺激產生的蛋白質抑制因子histone H4 有關。
並列摘要
Colchicine damages hair follicles and causes anagen- or telogen-hair loss, a severe side effect during treatment. It is the dermal papilla cells in hair follicle control its development and hair cycling. The secreted factors involved in the colchicine-induced hair loss have not been fully elucidated so far. Here, we purified and analyzed the secretomes of colchicine-treated and -untreated dermal papilla cells using the in vitro primary culture of dermal papilla cell. A heat-labile, trypsin-sensitive protein in the secretome of dermal papilla cells treated with 2 μM of colchicine for 6 hours inhibited 18.5 % growth and reduced 74.1 % alkaline phosphatase activity of dermal papilla cells. After 2-DE separation and analysis, 17 of 26 proteins have higher than 2-fold level after colchicine treatment. Three protein spots were matched in Swissprot database searching with statistical significance (p<0.05). Using LC-ESI MS/MS and Mascot on-line database searching, we identified this growth-inhibition protein as histone H4 (gi|4505821), which was further validated by a Western blot with an anti-histone H4 antibody. Our results indicate that colchicine stimulates dermal papilla cells to secrete a protein, further influencing the protein expression of dermal papilla cells.