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研究生:陳惠琬
研究生(外文):Hui-Wan Chen
論文名稱:纖維蛋白原與短鏈透明質酸還原端偶合物之合成與鑑定
論文名稱(外文):Synthesis and characterization of fibrinogen coupled to the reducing-end of short chain hyaluronate
指導教授:王盈錦
指導教授(外文):Yng-Jiin Wang
學位類別:碩士
校院名稱:國立陽明大學
系所名稱:醫學工程研究所
學門:工程學門
學類:生醫工程學類
論文種類:學術論文
論文出版年:2004
畢業學年度:92
語文別:中文
論文頁數:99
中文關鍵詞:纖維蛋白原透明質酸還原端透明質酸寡醣
外文關鍵詞:fibrinogenhyaluronic acidreducing endhyaluronan oligomer
相關次數:
  • 被引用被引用:2
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摘要
在人體傷口癒合的不同階段中,纖維蛋白及短鏈HA(short chain hyaluronate,sHA)分別扮演細胞模板及提供細胞訊號的功能。本研究以此作為理論基礎,於纖維蛋白原中引入sHA,在保留纖維蛋白原可凝膠的特性,並同時具有sHA能提供細胞訊號優點的考量下,選擇以還原胺法偶合sHA及纖維蛋白原,期望能開發出一具有細胞載體應用潛力的新材料。
纖維蛋白原與sHA經還原端偶合後之產物(稱為SXF,代表sHA
(S)與纖維蛋白原(F)之莫耳比為X:1)所得SXF之蛋白質濃度約分佈於6-8 mg/ml,產率約介於60-70%,其接枝比由低至高依序排列為S30F < S40F < S50F < S60F < S70F。隨sHA接枝率的增加,sXF之自由胺基濃度及GPC滯留時間將逐漸下降,其自由胺基含量(Free amino group : F molar ratio)由高至低分別為S30F(1084.55)> S40F(932.00)> S50F(915.25)> S60F(880.53)> S70F(760.85)。平均粒徑則隨接枝率上升而增加,其由小至大分別為S30F(38.5 nm)< S40F(42.7 nm)< S50F(46.0 nm)< S60F(81.4 nm)< S70F(97.4 nm)。
電泳分析的結果顯示,SXF三個次單元的分子量皆明顯較纖維蛋白原(α:63.6 kD;β:59.3 kD;γ:48 kD)來的高,且其值隨分子量的增加而增加。由此結果可推論,纖維蛋白原的三個次單元中皆存在sHA的接枝。此外,SXF的可凝結蛋白含量於接枝比4-6間存在一明顯轉折,也就是說當纖維蛋白原上的lysine被改質數目超過6個時,將導致凝結能力的明顯下降。SXF隨接枝比的增加,其CP值的變化如下:S30F(接枝比:1.27,CP:94.46%)> S40F(接枝比:4.20,CP:82.61%)> S50F(接枝比:6.17,CP:55.54%)> S60F(接枝比:11.20,50.23%)> S70F(接枝比:15.98,CP:45.11%)。
降解結果顯示,SXF仍具被trypsin降解的能力,然而其降解產物與纖維蛋白原並不相同,其降解產物之分子量皆較纖維蛋白來得高。
SEM觀察的結果發現,經sHA改質後的纖維蛋白,表面皆呈現凹凸不平的現象,且明顯覆蓋一層物質,其覆蓋量隨接枝比的增加而增加。纖維側面聚集現象亦有增加的趨勢,其中以S50F最為明顯。且sHA改質後,纖維蛋白之纖維末端有逐漸變細的現象,並以S40F最為顯著。此外,纖維直徑及孔隙度於接枝比4-6間亦發現存在一轉折點。S30F(接枝比:1.27)與S40F(接枝比:4.2)結構與纖維蛋白原無明顯差異,而S50F(接枝比:6.17)則明顯與其不同,此點與由CP結果所得之改質臨界點結果相符。
綜合實驗結果可知,SXF低於接枝比6,保留了纖維蛋白的結構及凝結能力。而sHA的引入,使其具有提供細胞生長訊號的特點,於細胞載體應用上的有相當之潛力,故未來應朝向細胞培養及實際應用方向繼續探討。然而當接枝比增加至6以上,其凝結能力明顯下降,相對延長凝結時間,對於需快速凝膠特性的應用上,可能需要藉由增加thrombin使用量,或將之與纖維蛋白原混合,始能予以改善,這也是未來研究應進行的方向。
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