- 學位論文
N-乙醯基半胱氨酸於傷口癒合生物效應與影響之探討
The Biological Effects of N-Acetylcysteine on Promoting Wound Healing
摘要
N-乙醯基半胱氨酸（N-Acetylcysteine，Nac）是一種抗氧化劑，目前臨床上的使用，僅有口服和注射兩種劑型。本研究運用Nac，以體外（in vitro）和體內（in vivo）的方式，局部治療燒燙傷口，以研究其作用機制。 在體外方式的研究中，我們把人類表皮纖維母細胞CCD-966SK經過Nac處理，然後監控其穀胱甘胺酸（Glutathione）數量、細胞增殖情況、移動、抓傷癒合活動，並且監控上皮化（epithelialization）相關蛋白質、基質金屬蛋白酶-1（matrix metalloproteinases-1，MMP-1）和與MMP-1表現相關的蛋白質。不同的Nac濃度（0.1、0.5和1.0 mM）均會增加CCD-966SK細胞的麩胱甘肽數量、細胞存活率、抓傷癒合活性和移動能力，增加程度和劑量相關。以1.0 mM的Nac 處理CCD-966SK細胞24小時，MMP-1表現會顯著增加。CCD-966SK細胞經Nac誘導後，磷脂酸肌醇3-激酶（phosphatidylinositol 3-kinase，PI3K）表現量隨Nac劑量增加(0.1 mM 、0.5 mM 、1.0 mM)相較於對照組，分別增加12％、31％和74％。此外，CCD-966SK細胞經1.0 mM Nac誘導一天後，相較於對照組，磷酸化蛋白質激酶C（phospho-PKC，p-PKC)、PKC、磷酸化詹納斯激酶(phospho-Jak，p-Jak1)、Jak1、磷酸化轉錄因子3的訊號傳導和活化蛋白（phospho- signal transducer and activator of transcription 3，p-Stat 3)、Stat 3、c-Fos和Jun蛋白表現量分別增加了263%、44%、206%、34%、316%、169%、68%、及 64%，增加程度和劑量相關，不過細胞外訊號調節激酶1和2（extracellular signal-regulated protein kinases 1 and 2，Erk1/2）則沒有增加現象。此外，透過PKC/Stat3訊號傳導路徑，Nac會引發MMP-1的膠原性（collagenous）表現。 在體內方式的研究中，使用燒燙傷口癒合的大鼠模型來評估Nac的刺激活性和組織病理學效果，各組的再生表皮百分率如下(%)：(a) untreated control, 8.60 ± 3.33; (b) positive control, 14.90 ± 2.56; (c) vaseline, 10.94 ± 3.19; (d) 0.1% Nac, 10.74 ± 1.47; (e) 0.5% Nac, 14.47 ± 0.80; (f) 3.0% Nac, 19.02 ± 2.85。相較於untreated control組，0.5％ Nac和3.0％ Nac組的再生表皮百分比均顯著高於對照組（分別為P<0.05及P<0.01）。而且，相較於vaseline 和positive control組，3.0％ Nac組再生表皮百分比均顯著高於這兩組，P值均<0.05。結果發現，3.0% Nac處理過的傷口，再上皮化（re-epithelialization）的情況較佳。 我們的結果證實，Nac可能可以提升傷口癒合活性，對於促進燒燙傷口的癒合，是一項具有前景的藥物。
並列摘要
N-Acetylcysteine (Nac) is an antioxidant administered in both oral and injectable forms in clinics. Here we used Nac topically on treating burn wounds in vitro and in vivo to investigate the mechanisms. In vitro, we detected glutathione levels, cell proliferation, migration, scratch-wound healing activities and the epithelialization-related proteins; matrix metalloproteinase-1 (MMP-1) and proteins involved in the formation pathway of MMP-1 of CCD-966SK cells treated with Nac. Various Nac concentrations (0.1, 0.5, and 1.0 mM) increased glutathione levels, cell viability, scratch-wound healing activities and migration abilities of CCD-966SK cells in a dose-dependent manner. MMP-1 expression of CCD-966SK cells treated with 1.0 mM Nac for 24 hrs was significantly increased. Levels of PI3K, PKC, Jak1, Stat3, c-Fos and Jun, but not Erk1/2, also significantly increased with dose dependence compared to the control. In addition, Nac induced collagenous expressions of MMP-1 via PKC/Stat3 signaling pathway. In vivo, the burn wound healing model of rats was applied to assess the stimulating activity and the histopathologic effects of Nac, with 3.0% Nac-treated wounds being found to show better characteristics on re-epithelialization. Our results demonstrated Nac potentially promoted wound healing activity. Nac may be a promising drug for accelerating burn wound healing.