- 學位論文
類薑黃素(curcuminoids) 與Seprafilm®在小鼠傷口癒合模式之研究
The animal study of curcuminoids and Seprafilm® in wound healing
摘要
傷口的癒合過程是一個很複雜的生物機制。當組織受到損傷後，在修復過程中依循此機制可完整地完成傷口癒合。在傷口癒合過程中包括：發炎反應、細胞增生，其次為新的組織再生，且包含了血管的增生、細胞外基質合成、膠原蛋白的重組以及上皮細胞的再生。在本論文中使用天然植物所萃取的類薑黃素(Curcuminoids)作為實驗藥物之一；探討類薑黃素對於促進傷口癒合的療效與可能的調控機轉。而類薑黃主要的成份為薑黃素(Curcumin)；去甲氧基薑黃素(Demethoxycurcumin)和二甲氧基薑黃素(Bisdemethoxycurcumin)。目前已知它具有抗發炎、抗氧化、清除自由基、抗癌、抑制感染的活動力及膠原蛋白生成作用。 防沾黏膜Seprafilm®在臨床上應用於胃腸或骨盆剖腹部手術患者，且減少手術後沾黏障礙。防沾黏薄膜主要成分為玻尿酸(Sodium Hyaluronate)和羧甲基纖维素(Carboxymethylcelluluse)混合成膠紙狀，臨床上報告提出它可以防止組織沾黏與協助組織修復。當傷口修復過程中有許多細胞訊息參與其中，如纖維化因子(Fibrotic factor)與生長因子(Growth factor)是最廣泛作為探討與研究的對象，這些因子結合在纖維母細胞的細胞膜上特定的接受器，開始訊息傳遞啟動細胞內功能性基因表現。在本論文研究中將探討類薑黃素及防沾黏膜Seprafilm®，在小鼠傷口癒合過程中調控轉化生長因子-β (Transforming growth factor-β; TGF-β)、熱休克蛋白(Heat shock protein 47; Hsp-47)以及第一型膠原蛋白 (Collagen I)作用機制，並以zymography在傷口中分析MMP-2及MMP-9的作用與相關機轉。在本實驗中，持續六天分別塗抹不同劑量類薑黃素及防沾黏膜Seprafilm®在小鼠背部傷口，並每天以影像記錄傷口癒合情形，於第七天將傷口週邊組織取下並以組織染色法及西方墨點法(Western blotting)進行分析研究。結果後得知；類薑黃素對於傷口癒合外觀並無明顯改善之功效。但在西方墨點法的結果中，可以發現類薑黃素分別在不同劑量(0.1mg/kg及0.5mg/kg)，對於誘導一氧化氮合成酵素 (Inducible nitric oxide synthase; iNOS)，轉化生長因子-β，熱休克蛋白及第一型膠原蛋白的生成上與控制組比較並有顯著的差異性。利用zymography分析傷口MMPs中的MMP-2與MMP-9酵素還原酶的活性， Seprafilm®與未治療組MMP-9則有還原效素活性。Seprafilm®與未治療組比較在一氧化氮合成酵素則明顯的具有統計上的差異。其實驗結果Seprafilm®與類薑黃素比較，類薑黃素會促使膠原蛋白合成且促進傷口癒合。
並列摘要
Wound healing is a complex biological process. Wound healing consists of an orderly progression of events that re-establish the integrity of the damaged tissue, including inflammation, cell migration, angiogenesis, extracellular matrix synthesis, collagen deposition and re-epithelialization. There are three major stages in the wound healing process; the first stage is the inflammatory response, then proliferation and finally tissue regeneration. The present investigation was undertaken to determine the role of curcuminoids and Seprafilm on changes in collagen characteristics and antioxidant property during cutaneous wound healing .Turmeric (Curcuminoids), an herb isolated from the rhizome of the plant, called Curcuma Longa. Interest in this herb has grown in recent years because of its putative beneficial health effects, including antioxidant, anti-inflammatory and cancer chemopreventive actions. The curcuminoids products contain a mixture of Curcumin, the primary ingredient, with minor components of demethoxycurcumin (DMC) and bisdemethoxycurcumin (bDMC). Seprafilm® Adhesion Barrier serves as a temporary bioresorbable barrier separating apposing tissue surfaces. The physical presence of the membrane separates adhesiogenic tissue while the normal tissue repair process takes place. The wound repair process have focused on signal molecules such as cytokines and growth factors. It is well known that these factors bind to specific receptors on the membrane of fibroblasts, which initiates a signal cascade, leading to specific cellular functions such as cell locomotion. The transforming growth factor-β (TGF-β) also promotes the fibroblast proliferation and collagen deposition. The heat shock protein 47 (HSP-47) is a collagen type I that has been shown to play a major role during the processing and/or secretion of procollagen. In this study, the curcuminoids were dissolved in 95% ethanol and the ICR mice were treated daily for 6 days at dosages (0.1 and 0.5 mg/kg). Initially, various doses of curcuminoids were administered topically daily in ICR mice to evaluate its wound healing capacity. On the 7th day post wounding, the ICR mice were sacrificed and protein expressions of Inducible Nitric oxide synthase (iNOS), transforming growth factor-β, heat shock protein 47, collagen I were determined by western blotting. The MMPs expressions of MMP-2 and MMP-9 were determined by zymography (Gelatin-substrate Zymography). Our results suggest that curcuminoids was found to improve all the phases of wound repair, including collagen synthesis.