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研究生:郭奕炆
研究生(外文):Yi-Wen Kuo
論文名稱:以雷射誘導內皮細胞損傷及大鼠血管?生探討納豆激?之功能
論文名稱(外文):Using laser induced injury of the endothelial cells and the rat’s blood vessel thickening to study the effects of nattokinase
指導教授:吳彰哲
指導教授(外文):Chang-Jer Wu
學位類別:碩士
校院名稱:國立臺灣海洋大學
系所名稱:食品科學系
學門:農業科學學門
學類:食品科學類
論文種類:學術論文
論文出版年:2009
畢業學年度:97
語文別:中文
論文頁數:63
中文關鍵詞:雷射納豆機?內皮細胞
外文關鍵詞:lasernattokinaseendothelial cells
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台灣逐漸邁入高齡化社會,其中一個長久以來威脅人類壽命的疾病-「心腦血管相關的疾病」。根據我國衛生署統計資料,台灣地區於民國95年死於腦血管疾病及心臟相關疾病各約為12596人與12283人,各居該年十大死因的第二位及第三位。近年來研究指出,納豆具有預防與治療心臟血管疾病,並可恢復疲勞與抗腳氣病的功能。故本論文將利用內皮細胞及大鼠動物實驗來探討納豆萃取物及納豆激?抑制血栓的功效。首先,利用在血纖維蛋白平板試驗比較相同攪拌時間、不同溫度培養條件下的納豆萃取物及純納豆激?在血栓培養基上所產生抑制血栓透明環之能力。結果顯示,在27℃攪拌2小時的納豆萃取物所產生抑制血栓透明環的效果最好。在細胞實驗中,內皮細胞(endothelial cells)加入不同濃度的染劑及有無照光反應試驗後,再利用MTS試驗測試其細胞存活率。結果顯示,染劑在0.125 mg/ml的濃度下單獨作用是無毒性。但在有照光時,內皮細胞的細胞存活率顯著的下降,顯示光照會使染劑激發而造成內皮細胞的死亡。納豆激?及納豆萃取物對內皮細胞之毒性試驗中,納豆激?在0.06 mg/ml以上具有毒性,但納豆萃取物在0.5 mg/ml下是不具細胞毒性。而從治療組看,納豆萃取物則有顯著減緩細胞凋亡的能力出現,所以可以得知納豆萃取物對於細胞有治療的能力。從內皮細胞的發炎相關基因來探討,IL-6、IL-1β、TNF-α在添加納豆激?和納豆萃取物之後和對照組相比之下都有顯著的下降。在動物實驗中,利用染劑和雷射光刺激大鼠造成頸動脈來建立血栓動物模式。結果顯示,大鼠在染劑和雷射光刺激之後的第28天,其頸動脈血管增生效果最為顯著。再利用螢光染色及共軛焦顯微鏡觀察,可以發現血管壁上的內皮細胞損傷已修補完全,但血管平滑肌的部分卻有顯著的增厚。從數據和照片顯示餵食納豆激?和納豆萃取物的大鼠血管有顯著減緩增生的能力。綜合上述的研究成果,顯示納豆激?和納豆萃取物可以用於發展成預防心血管疾病之保健食品。
Taiwan is now getting to the older society; the cardiovascular illness will menace the human life. According to the research of the department of healthy, the brain-vascular illness and cardiopathy are the second and third leading cause of death in Taiwan at 2006, respectively. Recently, natto have shown the ability to prevent and cure from the cardiovascular illness. In this thesis, the cardiovascular prevention effects of nattokinase and natto-extracts will be evaluated by the laser induced injury of the endothelial cells and the rat thrombosis model. First, the fibrinolytic activity of the purified nattokinase and natto-extracts were determined by the clear zone size of fibrin plate. The result showed that the purified nattokinase got higher clear zone size than the natto-extracts from two hours incubation under normal atmospheric temperature. In vitro studies, we used MTS assay to study the cytotoxicity of the dye and laser treatment on the endothelial cells. The data showed that the rose bengal dye on 0.125 mg/ml alone did not causes the death of endothelial cells, but the survival rate of endothelial cells would reduce under the combined treatment of dye and laser. The toxicity concentration of nattokinase and natto-extracts were above 0.06 mg/ml and 0.5 mg/ml, respectively. Natto-extracts could reduce the apoptosis of endothelial cells induced by the combined treatment of dye and laser. The data also showed that nattokinase and natto-extracts could significantly reduce the inflammation gene (L-6, IL-1β and TNF-α) expression of endothelial cells induced by the combined treatment of dye and laser. In animal studies, we also used the combined treatment of dye and laser to induce endothelial injury of the neck artery of WKY rat. After 28 days of photochemically injury, the neck artery of WKY rat was significantly observed thickening by light photomicrographs. From confocal scanning laser microscopy, the endothelial cells of the blood vessel wall were damaged and repaired, and the thickness of the smooth muscle of blood vessel was observably increased. Treatment of nattokinase and natto-extracts could decrease the thickness of the smooth muscle of blood vessel in photochemically injure WKY rat. These results indicate that nattokinase and natto-extracts may be used as a health food supplement to prevent cardiovascular illness.
目 錄
目錄……………………………………………………………….……Ⅰ表目錄…………………………………………………………….……Ⅳ
圖目錄…………………………………………………………….……Ⅳ
附錄目錄…………………………………………………………….…Ⅵ
中文摘要…………………………………………………………….…Ⅶ英文摘要…………………………………………………………….…Ⅸ
壹、文獻回顧………………………………………………………..… 1
一、心血管疾病………………………………………………….…1
1、心血管疾病之種類……………………………………….…..1
2、血栓的形成……………………………………………….…. 5
3、常用市售藥物溶解血栓……………………………………...6
二、雷射器件及其特性……………………………………….…....6
三、納豆的介紹……………………………………………….…....8
1、納豆的歷史……………………………………………….…. 8
2、納豆的種類及製程方法………………………………..…….9
3、納豆中所含物質……………………………………………...10
3.1.維生素B1、B6、K及菸鹼酸…………………………..10
3.2.異黃酮及皂角甘………………………………………..10
3.3.麩胺酸…………………………………………………..10
3.4.卵磷脂…………………………………………………..11
4、納豆激?………………………………………………………11
5、納豆激?溶解血栓之實驗……………………………………12
四、研究目的…………………………………………………………...13
參、實驗材料與研究方法……………………………………………...13
一、材料……………………………………………………………13
二、實驗器材與藥品………………………………………………14
1. 分析試劑……………………………………………………14
肆、實驗分析方法……………………………………..………………15
1. 納豆菌株培養與保存…………………………….…..….…15
2.納豆萃取物之製備………………………………….…….…15
3. 納豆激?及納豆萃取物活性之測試…………………….…16
3.1 血栓培養皿之製備…………………………………….16
3.2 探討納豆激?及納豆萃取物對血栓分解?活性之測試……………………………………………………………16
3.3 探討納豆萃取物對血栓分解?活性分析……….……16

4. 抗氧化能力測試…………………………………….........…17
4.1 清除α,α-diphenyl-β-picryhydrazyl (DPPH)自由基能力之測定…………………………………………………………18
4.2清除超氧陰離子能力之測定…………………………..18
4.3螯合亞鐵離子能力之測定……………………………..20
5、納豆激?及納豆萃取物減緩大白鼠頸動脈內皮細胞凋亡之探討…………………………………………………….………21
5.1 建立內皮細胞體外雷射照射模式……………..…...…21
5.2 探討納豆激?及納豆萃取物對內皮細胞效果之影響
…………………………….……………………………...…25
6、納豆激?及納豆萃取物抑制大白鼠頸動脈血管增生模式
…………………………….……………………………………25
6.1 建立雷射誘發大白鼠頸動脈血管增生動物模式…….25
6.2 探討納豆激?及納豆萃取物對血管增生減緩效果之影響…………….………………………………..…….…26
伍、結果…………….…….………………………….…………...……28
陸、討論…………….…….……………………………….……...……32
柒、結論………….……………………………………….……........…37
捌、參考文獻………….……………………………………...……..…39





表 目 錄
表一、納豆激?之血纖維蛋白平板試驗。……………..………..…44
表二、納豆萃取物之血纖維蛋白平板試驗。…………..………...…45
表三、納豆萃取物凍乾粉之血纖維蛋白平板試驗。………….....…46
表四、納豆萃取物及納豆激?之DPPH 自由基之清除效應、超氧歧 化?及螯合亞鐵離子能力之測定。…………….…….…..…47

圖 目 錄
圖一、納豆萃取物凍乾粉之血栓分解?(FE)活性變化。.….…….…48
圖二、內皮細胞在不同劑量的rose bengal dye中,照green laser 和不照green laser的細胞存活率。…………….………….…..….…49
圖三、內皮細胞在劑量0.125 mg/ml rose bengal dye中,不同雷射照射時間點的細胞存活率。…………….……………..……….……..50
圖四、不同劑量的納豆激?(A)及納豆萃取物(B)對內皮細胞之毒性試驗。…………….………………………………..……….…..…..51
圖五、在染劑照光刺激下,加入相同劑量下的納豆激?及納豆萃取物對內皮細胞預防及治療的細胞存活率。…………….….…..…52

圖六、即時聚合?連鎖反應(Real-time polymerase chain reaction) 加入納豆激?及納豆萃取物經由染劑照光刺激後分析內皮細胞發炎和黏著因子相關基因表現的情形。………………...….…..…53
圖七、Green laser照射雷射10分鐘及rose bengal dye劑量60 mg/kg/c.c,利用光學顯微鏡觀察不同時間點誘導之大鼠動脈血管管壁增生狀況。……………………..……………….………54
圖八、Green laser照射雷射10分鐘及rose bengal dye劑量60 mg/kg/c.c,28天後冷凍切片,利用HE染色觀察大鼠動脈管壁增生狀況。(A) control組,(B)納豆激?餵食組,(C)納豆萃取物。….………55
圖九、Green laser照射雷射10分鐘及rose bengal dye劑量60 mg/kg/c.c,利用光學顯微鏡觀察餵食納豆激?組和納豆萃取物組減緩之大鼠動脈血管管壁增生狀況。……………...………56
圖十、Green laser照射雷射10分鐘及rose bengal dye劑量60 mg/kg/c.c, 28天後冷凍切片,利用共軛焦顯微鏡觀察大鼠動脈管壁增生狀況。(A) 內皮細胞,(B)平滑肌,(C)細胞核,(D)影像重疊。……………………..……………………….………57
圖十一、Green laser照射雷射10分鐘及rose bengal dye劑量60 mg/kg/c.c,餵食納豆激? 28天後冷凍切片,利用共軛焦顯微鏡觀察大鼠動脈管壁增生狀況。(A) 內皮細胞,(B)平滑肌,(C)細胞核,(D)影像重疊。…..…………………………...………58
圖十二、Green laser照射雷射10分鐘及rose bengal dye劑量60 mg/kg/c.c,餵食納豆萃取物28天後冷凍切片,利用共軛焦顯微鏡觀察大鼠動脈管壁增生狀況。(A) 內皮細胞,(B)平滑肌,(C)細胞核,(D)影像重疊。………………………………..………59
附 錄 目 錄
附錄表一、慣用之血栓溶解劑之比較。………………..……………60
附錄圖一、納豆激?的構造。………………………………..………61
附錄圖二 納豆激?3D結構式及活性位置。……………..….……62
附錄圖三、納豆激?分解血栓原理。……………………..…………63
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