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系統識別號 U0002-2407200815141700
中文論文名稱 咖啡因干擾斑馬魚胚胎之血管形成
英文論文名稱 Caffeine Treatment Disturbs the Angiogenesis of Zebrafish Larvae
校院名稱 淡江大學
系所名稱(中) 生命科學研究所碩士班
系所名稱(英) Graduate Institute of Life Sciences
學年度 96
學期 2
出版年 97
研究生中文姓名 葉建宏
研究生英文姓名 Chien-hung Yeh
學號 695180033
學位類別 碩士
語文別 中文
口試日期 2008-07-22
論文頁數 60頁
口試委員 指導教授-陳曜鴻
委員-蔡懷楨
委員-蘇銘燦
中文關鍵字 咖啡因  斑馬魚  脈管生成  血管新生 
英文關鍵字 Caffeine  Zebrafish  vasculogenesis  angiogenesis 
學科別分類 學科別醫學與生命科學生物學
中文摘要 咖啡因是一種我們日常生活中,隨時都可能食用到或接觸的物質。在醫學研究上也有使用咖啡因可保護肝臟、治療哮喘、減低罹癌機率等正面報導,同樣的又有引發心臟病、骨質疏鬆或者孕婦流產機率增高等負面訊息。因此,咖啡因對於人體是否有害爭論從未斷過。基於咖啡因對胚胎發育時期血管形成的影響沒有研究報導,所以本論文利用血管內皮細胞發出綠螢光的基因轉殖斑馬魚 Tg(fli1:egfp),來觀察咖啡因對斑馬魚胚胎血管生成的影響。將不同斑馬魚胚胎浸泡於不同濃度(0、150、250、350及500 ppm)咖啡因,都可發現咖啡因並不會影響斑馬魚胚胎脈管生成(vasculogenesis)。接著利用統計20節斑馬魚胚胎背縱軸血管 (dorsal longitudinal vessels, DLAV)之發育完成數做比較分析。結果未經咖啡因處理的DLAV數(19.42±0.13)與浸泡不同濃度(150、250、350及500 ppm)咖啡因之DLAV數(13.52±0.34%、8.18±0.30%、4.65±0.28% 及2.82±0.22%)相比較,發現確實影響斑馬魚胚胎的血管新生(angiogenesis),且與咖啡因的濃度成正比關係。浸泡咖啡因斑馬魚胚胎其即時定量PCR分析值,相對於未經咖啡因處理得到cdh5下降至0.36倍,而cmlc2、flt1及nrp1a1的值是上升的,依次為4.62倍、2.44倍及6.42倍,可以確實證明咖啡因會透過影響這些基因而干擾斑馬魚胚胎之血管形成。
英文摘要 Caffeine is a typical purine alkaloid that is produced in a variety of plants, including coffee, tea, kola nuts, guarana berries and cocoa beans. In medical applications, caffeine can be used as an antagonist to the adenosine receptor for regulating the contraction of blood vessels. Epidemiological studies have described the exposure of women to caffeine during pregnancy, as well as the occurrence of congenital malformations and fetal growth retardation. It might be assumed that caffeine treatment affects blood vessel formation. However, whether caffeine treatment affects vasculogenesis and/or angiogenesis during early embryonic development are unclear. Here, we used transgenic zebrafish Tg(fli1:egfp) as a model to study the caffeine-induced toxicity of vasculogenesis/angiogenesis. We soaked the embryos into different concentrations of caffeine, and found that caffeine treatment did not affect the vasculogenesis. With regard to angiogenesis, we analysed the development of dorsal longitudinal vessels in 20 somites. Results showed that caffeine-treated embryos had less dorsal longitudinal vessels compared to vehicle-control (150 ppm ~ 500 ppm 13.52±0.34 ~ 2.82±0.22;vehicle-control:19.42±0.13). On the basis of this observation, we suggested that caffeine treatment disturbs the angiogenesis of zebrafish embryos. Furthermore, quantitative polymerase chain reaction was carried out to check the expression level of cdh5, cmlc2, flt1, and nrp1a1. We found that cdh5 expression decreased to 0.36 folds, but cmlc2, flt1 and nrp1a1 increased to 4.62, 2.44 and 6.42 folds, respectively. Our conclusion is that; caffeine affected cdh5, cmlc2, flt1, and nrp1a1 genes and consequently disburbs the angiogenesis of zebrafish
larva.
論文目次 目 錄

中 文 摘 要------------------------------------------I
英 文 摘 要-----------------------------------------II
目 錄----------------------------------------III
圖 表 目 錄-----------------------------------------IV
前 言------------------------------------------1
材料與方法-----------------------------------------12
結 果-----------------------------------------20
討 論-----------------------------------------31
參 考 文 獻-----------------------------------------38
圖 表-----------------------------------------42
名 詞 對 照-----------------------------------------52
附 錄-----------------------------------------55




圖 表 目 錄

Fig. 1. 斑馬魚在胚胎時期血管發育-----------------------------42
Fig. 2. 咖啡因對不同時期斑馬魚胚胎血管發育之影響-------------43
Fig. 3. Method II (12~36 hpf) 觀察不同濃度咖啡因下斑馬魚
胚胎在24 hpf 及36 hpf的血管形成----------------------44
Fig. 4. Method III (6~30 hpf) 觀察斑馬魚胚胎在10 hpf 時,
血管內皮細胞的前驅細胞聚集---------------------------45
Fig. 5. Method II(12~36 hpf)在 36及60 hpf 形成 DLAV
的統計圖表-------------------------------------------46
Fig. 6. Method II(12~36 hpf)在 36及60 hpf 形成 DLAV
的缺陷程度分布---------------------------------------47
Fig. 7. Method II、IV及V在60 hpf 時之DLAV -----------------48
Fig. 8. Method IV(24~36 hpf)在 36及60 hpf 形成 DLAV
的統計及缺陷程度分布圖表-----------------------------49
Fig. 9. Method V(12~60 hpf)在 36及60 hpf 形成 DLAV
的統計及缺陷程度分布圖-------------------------------50
Fig. 10. cdh5、cmlc2、flt1 及 nrp1a1 之 Q-PCR 分析------------51

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