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研究生:陳建成
研究生(外文):Chen,Chinecheng
論文名稱:探討氧氣濃度對神經脊幹細胞增殖,凋亡及遷移的影響
論文名稱(外文):Effects of Oxygen Levels on the Proliferation, Apoptosis and Migration of Neural Crest Stem Cells.
指導教授:陳怡潓陳怡潓引用關係
指導教授(外文):Chen,Yihui
口試委員:陳怡潓,梁章敏,蕭仁傑
口試委員(外文):Chen,Yihui,Liang ,Changmin ,Shiao,Jenchieh
口試日期:2012-05-18
學位類別:碩士
校院名稱:國防醫學院
系所名稱:海底醫學研究所
學門:醫藥衛生學門
學類:醫學學類
論文種類:學術論文
論文出版年:2012
畢業學年度:100
語文別:中文
論文頁數:55
中文關鍵詞:神經脊幹細胞
外文關鍵詞:Neural Crest Stem Cells
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幹細胞治療是指輸入非藥物性的幹細胞到體內,藉由幹細胞的特性:增強幹細胞對組織細胞的再生和修復的生物能力,能重新再造身體老化器官之組織結構,進而修復不健康的身體,使各器官的組織正常化。目前在臨床上已有用來治療癌症、中風、糖尿病等。在高氧方面,許多病都需要用到高氧治療,例如:一氧化碳中毒、傷口的修復等。但是在進行高氧治療的同時,也提升了體內幹細胞接觸到更濃的氧氣濃度,那是否會影響到幹細胞的生理現象,尚未有明確的研究。目前許多研究已指出體內分佈於各器官和組織的缺氧環境對於防止成體幹細胞的分化並維持其休眠(quiescence)與自我更新(self-renewal)是不可或缺的。在高氧也有研究指出幹細胞會因為活性氧物種自由基(ROS)過高而凋亡(apoptosis),也有文獻指出高氧會促使幹細胞的遷移(migration)。而除了眾所皆知的骨髓間質幹細胞(bone marrow mesenchymal stem cells)外,近年來的研究發現位於成人的毛囊及齒囊內的神經脊幹細胞(neural crest stem cells)具有可取代胚胎幹細胞的功能性(pluripotency)。然而目前尚不清楚氧氣濃度對於神經脊幹細胞的影響。因此本論文研究旨在探討氧氣濃度是否會影響由成體毛囊內取得之神經脊幹細胞的增殖、遷移及凋亡等生理現象。
實驗中分為三個部分,以20%的氧氣為控制組。在第一部分我們先確立了不同的氧氣濃度(1%、20%、80% O2)會造成神經脊幹細胞生長數目有所不同。第二部分我們使用的EdU和TUNEL鑑別神經脊幹細胞的增殖與凋亡。第三部分我們用免疫染色法鑑別遷移的標定蛋白(cortactin、actin、CXCR4)。
結果發現培養在高氧組的細胞總數有較低的趨勢,而藉由EdU的結果也可以看到低氧組的增殖率與控制組沒有差別,在高氧組的結果可以看到明顯的低於控制組。以TUNEL的結果可以看到低氧組的細胞凋亡與控制組沒有差別,在高氧組可以看到凋亡數明顯的多於控制組。在遷移方面,標記蛋白的免疫染色顯示三個組別彼此之間沒有顯著差異。
綜合目前的發現,細胞給予低氧的情況下細胞總數會略微增加但增殖與凋亡都沒有統計上的意義,給予高氧的情況下細胞總數會明顯的降低,增殖率也會降低,凋亡率會增加。但是不同的氧氣濃度對於細胞的遷移沒有顯著的影響。

Stem cell therapy is to inject non-pharmacological stem cells into the human body and utilize the strong regenerative and repairing capacity of stem cells to renew and regenerate the tissue structures of damaged or aged organs, thus to repair the unhealthy body and normalize the tissues of each organ. Currently, stem cell therapy has been applied clinically to cure cancers, stroke, diabetes, etc.. As for hyperoxia, many diseases and symptoms must be cured by hyperoxia therapy, i.e., carbon monoxide poisoning and would healing, etc.. Hyperoxia therapy increases the oxygen concentration in the human body, but there has not been conclusive research result about the effect of high oxygen levels on the physiological behaviors of adult stem cells. To date many researches have indicated that the hypoxic environment in each organ and tissue of our human bodies is indispensible for preventing differentiation of adult stem cells as well as maintaining their quiescence and self-renewal. Under hyperoxic environment, previous studies have indicated that the high level of ROS (reactive oxygen species) increases stem cell apoptosis, and that hyperoxia can promote stem cell migration. In addition to the well-known bone marrow stem cells, in recent years it was found that neural crest stem cells isolated from adult hair follicles and dental follicles are almost as pluripotent as embryonic stem cells. However, currently it is not clear yet how the levels of oxygen affect the behaviors of neural crest stem cells. Therefore, my thesis research is aimed to investigate whether oxygen concentrations affect the proliferation, migration and apoptosis of neural crest stem cells isolated from adult hair follicle.
Our experiments used 20% oxygen as the control environment, and could be divided into three parts. In the first part, we confirmed that culturing under different oxygen concentrations (1%、20%、40% and 80% O2) led to different total cell numbers of neural crest stem cells. In the second part, we performed EdU and TUNEL staining to identify the proliferating and apoptotic neural crest stem cells, respectively. In the third part, we performed immunostaining to analyze expression of proteins involved in cell migration, including actin, cortactin and CXCR4.
We found that the total cell numbers of neural crest stem cells were significantly decreased under high oxygen concentrations (40% and 80% O2). The EdU staining showed that the proliferation rates of neural crest stem cells were comparable between hypoxic and normoxic culturing environments, whereas the proliferation rate was significantly lower under hyperoxia compared with that under normoxia. The TUNEL staining showed that the apoptosis rates of neural crest stem cells were comparable between hypoxic and normoxic culturing environments, whereas the apoptosis rate was significantly higher under hyperoxia compared with that under normoxia. As for the immunostaining with migration markers, we did not observe significant differences among hypoxic, normoxic and hyperoxic environments.
In summary, while culturing under hypoxia, the total number of neural crest stem cells has a small decrease without significant changes in both the proliferation and apoptosis rates. While culturing under hyperoxia, the total number and proliferation rate of neural crest stem cells both significantly decrease, while their apoptosis rate significantly increases. On the other hand, alteration of oxygen concentrations has no significant effect on neural crest stem cell migration.

中文摘要……………………………………………………………I
英文摘要……………………………………………………………III
緒論…………………………………………………………………第1頁
材料方法……………………………………………………………第11頁
實驗結果……………………………………………………………第20頁
討論…………………………………………………………………第26頁
參考文獻……………………………………………………………第32頁
表……………………………………………………………………第39頁
圖……………………………………………………………………第38頁

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