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研究生:黃雅君
研究生(外文):Ya-Chun Huang
論文名稱:新型血管新生因子(GinsenosideRg1與Re)與其在組織工程的應用
指導教授:宋信文
學位類別:碩士
校院名稱:國立清華大學
系所名稱:化學工程學系
學門:工程學門
學類:化學工程學類
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:66
中文關鍵詞:血管增生組織工程Ginsenoside Rg1Ginsenoside Re
相關次數:
  • 被引用被引用:2
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  • 下載下載:28
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摘要
在組織工程的研究裡,常遭遇到植入生物體內後的人工細胞外間質血管增生不足,造成遷入的細胞與新生的組織無法獲得充分的氧氣與養分。為了解決這項問題,文獻中曾有研究群在人工細胞外間質內加入生長因子,以促進其內的血管增生。目前用來當做促進血管增生的生長因子大部分都是蛋白質,其在生物體內的穩定性不佳,很容易失去其活性。為了克服這項問題,我們著手使用了從中藥〝人參〞裡,萃取純化出來的ginsenoside Rg1 (Rg1)與ginsenoside Re (Re)來當做新型的促進血管生長因子。
本研究主要分成三部份來進行。在第一和第二部分的實驗裡,我們以體外細胞培養測試的方式,分別評估了Rg1與Re對人類臍帶靜脈內皮細胞(HUVECs)的增殖、遷徙以及tube formation的影響。實驗裡,我們以在細胞培養基中未添加任何藥物當做空白對照組,而以在細胞培養基中加入bFGF當做正對照組。在本部份的實驗裡,我們證實了Rg1與Re具有與目前文獻上使用的血管生長因子(如bFGF)相同的特性,可以促進HUVECs增殖、遷徙以及tube formation的能力。同時我們也發現Rg1較親水,而Re則屬疏水性藥物。
在第三部分的體內實驗裡,我們以genipin交聯經醋酸膨潤與酵素處理的去細胞牛心包膜,當做人工細胞外間質(ECM),然後把Re以gelatin包覆在ECM (ECM/Re)內後植入老鼠背部,探討Re在生物體內對血管增生以及組織再生與修復的影響。Rg1的體內實驗探討已由本實驗室博士班學生 梁晃千學長完成。實驗裡的對照組為bFGF (ECM/bFGF),以及未包覆任何生長因子的ECM (ECM/ control)。在實驗設計上,我們分別於1星期與1個月後取樣,針對植入後人工細胞外間質內的血管增生數目、免疫反應、細胞遷入種類與滲入數目(infiltrated cell number)、組織修復時新生的細胞外間質組成以及變性溫度等加以評估。
實驗結果顯示,ECM/Re與ECM/bFGF在植入1星期後,老鼠的免疫細胞、內皮細胞以及紅血球都可以滲進多孔性結構的ECM內部。ECM/control內免疫細胞的數目明顯的比其他兩種試片來得多,但新生血管滲入包覆bFGF或Re的 ECM密度、深度以及hemoglobin含量,都明顯的比未包覆任何生長因子的ECM 來得多,這個現象顯示了包覆bFGF或Re的ECM可以明顯促進ECM內的血管增生。
植入1個月後,ECM/Re的外層孔洞結構內免疫細胞已經消失殆盡,取而代之的為宿主遷徙進來的纖維母細胞,以及其所分泌的結締組織和供應養分與氧氣的新生微血管。另外在ECM/bFGF的外層結構內也可以觀察到組織再生的情形,但仍有相當數目的免疫細胞存在,而ECM/control結構內組織再生的情形則較不明顯且免疫反應仍很強烈。另外ECM/Re在植入1個月後,其結構內新生血管密度與hemoglobin含量,皆明顯的比植入1星期後的量來得多。相對地,ECM/bFGF在植入1個月與1星期後的新生血管並沒有明顯的增加,而ECM/control內的血管增生情形並不顯著。此結果顯示了,Re在ECM內能夠持續有效的促進血管增生,而bFGF可能由於其穩定性不佳很快的失去活性,無法持續有效的促進ECM內的血管增生。
上述體外與體內實驗結果顯示,Rg1與Re皆為有效促進血管增生的新型生長因子,且可加速組織再生及修復的速率。未來我們可利用Rg1與Re親疏水性質的不同,分別應用於親水性與疏水性材料的包覆釋放。
目 錄
內容 頁數
摘要 I
目錄 III
圖索引 VI
圖索引---------------------------------------------------------------------------- X

第一章 緒論
1.1 組織工程 1
1.2 組織工程面臨的難題 1
1.3 新型血管新生因子 2
1.4 五加參皂苷Rg1 (Ginsenoside Rg1) 2
1.5 五加參皂苷Re (Ginsenoside Re) 3
1.6 血管新生機制 4
1.7 人工細胞外間質 5
1.8 去細胞生物組織 7
1.9 研究動機與目的 8

第二章 體外實驗—Ginsenoside Rg1
2.1 研究目的 10
2.2 材料與方法 10
2.2.1細胞培養(Cell Culture) 10
2.2.2 HUVECs增殖實驗 11
2.2.3 HUVECs遷徙實驗 11
2.2.4 HUVECs tube formation實驗 14
2.3 實驗結果與討論 15
2.3.1 Rg1對HUVECs之增殖作用 15
2.3.2 Rg1對HUVECs之遷徙作用 17
2.3.3 Rg1對HUVECs之tube formation作用 20
2.4 結論 22

第三章 體外實驗—Ginsenoside Re
3.1 研究目的 24
3.2 材料與方法 24
3.2.1 細胞培養(Cell Culture) 24
3.2.2 Re溶液的配置 24
3.2.3 HUVECs增殖實驗 24
3.2.4 HUVECs遷徙實驗 25
3.2.5 HUVECs tube formation實驗 25
3.3 實驗結果與討論 26
3.3.1 Re對HUVECs之增殖作用 26
3.3.2 Re對HUVECs之遷徙作用 28
3.3.3 Re對HUVECs之tube formation作用 29
3.4 結論 33

第四章 體內實驗—Ginsenoside Re
4.1 研究目的 35
4.2 實驗材料 35
4.2.1 ECM的製備 35
4.2.2 製備包覆bFGF或Re的ECM 37
4.3 實驗方法 39
4.3.1 ECM檢測 39
4.3.2 體內實驗 42
4.4 實驗結果與討論 46
4.4.1 ECM的製備 46
4.4.2 體內實驗 47
4.5 結論 59

參考文獻 60
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