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。

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.

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

Adams RH, Alitalo K, 2007. Molecular regulation of angiogenesis and lymphangiogenesis. Nat Rev Mol Cell Biol 8: 464-478.
Aldridge A, Aranda JV, Neims AH, 1979. Caffeine metabolism in the newborn. Clin Pharmacol Ther 25: 447-453.
Ashihara H, Crozier A, 2001. Caffeine: a well known but little mentioned compound in plant science. Trends Plant Sci 6: 407-413.
Blum Y, Belting HG, Ellertsdottir E, Herwig L, Luders F, Affolter M, 2008. Complex cell rearrangements during intersegmental vessel sprouting and vessel fusion in the zebrafish embryo. Dev Biol.
Bolton S, Null G, Troetel WM, 1982. The medical uses of garlic--fact and fiction. Am Pharm NS22: 40-43.
Carmeliet P, 2003. Blood vessels and nerves: common signals, pathways and diseases. Nat Rev Genet 4: 710-720.
Carmeliet P, Tessier-Lavigne M, 2005. Common mechanisms of nerve and blood vessel wiring. Nature 436: 193-200.
Chen YH, Huang YH, Wen CC, Wang YH, Chen WL, Chen LC, Tsay HJ, 2008. Movement disorder and neuromuscular change in zebrafish embryos after exposure to caffeine. Neurotoxicol Teratol.
Childs S, Chen JN, Garrity DM, Fishman MC, 2002. Patterning of angiogenesis in the zebrafish embryo. Development 129: 973-982.
Dassesse D, Ledent C, Parmentier M, Schiffmann SN, 2001. Acute and chronic caffeine administration differentially alters striatal gene expression in wild-type and adenosine A(2A) receptor-deficient mice. Synapse 42: 63-76.
Durbin L, Sordino P, Barrios A, Gering M, Thisse C, Thisse B, Brennan C, Green A, Wilson S, Holder N, 2000. Anteroposterior patterning is required within segments for somite boundary formation in developing zebrafish. Development 127: 1703-1713.
Ferrara N, 2004. Vascular endothelial growth factor: basic science and clinical progress. Endocr Rev 25: 581-611.
Ferrara N, Hillan KJ, Gerber HP, Novotny W, 2004. Discovery and development of bevacizumab, an anti-VEGF antibody for treating cancer. Nat Rev Drug Discov 3: 391-400.
Folkman J, Shing Y, 1992. Angiogenesis. J Biol Chem 267: 10931-10934.
Fouquet B, Weinstein BM, Serluca FC, Fishman MC, 1997. Vessel patterning in the embryo of the zebrafish: guidance by notochord. Dev Biol 183: 37-48.
Geretti E, Shimizu A, Klagsbrun M, 2008. Neuropilin structure governs VEGF and semaphorin binding and regulates angiogenesis. Angiogenesis 11: 31-39.
Gerhardt H, Golding M, Fruttiger M, Ruhrberg C, Lundkvist A, Abramsson A, Jeltsch M, Mitchell C, Alitalo K, Shima D, Betsholtz C, 2003. VEGF guides angiogenic sprouting utilizing endothelial tip cell filopodia. J Cell Biol 161: 1163-1177.
Hansel DE, Wilentz RE, Yeo CJ, Schulick RD, Montgomery E, Maitra A, 2004. Expression of neuropilin-1 in high-grade dysplasia, invasive cancer, and metastases of the human gastrointestinal tract. Am J Surg Pathol 28: 347-356.
Hollingsworth RG, Armstrong JW, Campbell E, 2002. Caffeine as a repellent for slugs and snails. Nature 417: 915-916.
Holmgren P, Norden-Pettersson L, Ahlner J, 2004. Caffeine fatalities--four case reports. Forensic Sci Int 139: 71-73.
Isogai S, Horiguchi M, Weinstein BM, 2001. The vascular anatomy of the developing zebrafish: an atlas of embryonic and early larval development. Dev Biol 230: 278-301.
Isogai S, Lawson ND, Torrealday S, Horiguchi M, Weinstein BM, 2003. Angiogenic network formation in the developing vertebrate trunk. Development 130: 5281-5290.
Kamei M, Saunders WB, Bayless KJ, Dye L, Davis GE, Weinstein BM, 2006. Endothelial tubes assemble from intracellular vacuoles in vivo. Nature 442: 453-456.
Kamijo Y, Soma K, Asari Y, Ohwada T, 1999. Severe rhabdomyolysis following massive ingestion of oolong tea: caffeine intoxication with coexisting hyponatremia. Vet Hum Toxicol 41: 381-383.
Kerrigan S, Lindsey T, 2005. Fatal caffeine overdose: two case reports. Forensic Sci Int 153: 67-69.
Kimmel CB, Ballard WW, Kimmel SR, Ullmann B, Schilling TF, 1995. Stages of embryonic development of the zebrafish. Dev Dyn 203: 253-310.
Lampugnani MG, Resnati M, Raiteri M, Pigott R, Pisacane A, Houen G, Ruco LP, Dejana E, 1992. A novel endothelial-specific membrane protein is a marker of cell-cell contacts. J Cell Biol 118: 1511-1522.
Larson JD, Wadman SA, Chen E, Kerley L, Clark KJ, Eide M, Lippert S, Nasevicius A, Ekker SC, Hackett PB, Essner JJ, 2004. Expression of VE-cadherin in zebrafish embryos: a new tool to evaluate vascular development. Dev Dyn 231: 204-213.
le Noble F, Fleury V, Pries A, Corvol P, Eichmann A, Reneman RS, 2005. Control of arterial branching morphogenesis in embryogenesis: go with the flow. Cardiovasc Res 65: 619-628.
le Noble F, Moyon D, Pardanaud L, Yuan L, Djonov V, Matthijsen R, Breant C, Fleury V, Eichmann A, 2004. Flow regulates arterial-venous differentiation in the chick embryo yolk sac. Development 131: 361-375.
Lee P, Goishi K, Davidson AJ, Mannix R, Zon L, Klagsbrun M, 2002. Neuropilin-1 is required for vascular development and is a mediator of VEGF-dependent angiogenesis in zebrafish. Proc Natl Acad Sci U S A 99: 10470-10475.
Meyer FP, Canzler E, Giers H, Walther H, 1991. [Time course of inhibition of caffeine elimination in response to the oral depot contraceptive agent Deposiston. Hormonal contraceptives and caffeine elimination]. Zentralbl Gynakol 113: 297-302.
Miao HQ, Klagsbrun M, 2000. Neuropilin is a mediator of angiogenesis. Cancer Metastasis Rev 19: 29-37.
Moffatt JD, Cocks TM, Page CP, 2004. Role of the epithelium and acetylcholine in mediating the contraction to 5-hydroxytryptamine in the mouse isolated trachea. Br J Pharmacol 141: 1159-1166.
Nathanson JA, 1984. Caffeine and related methylxanthines: possible naturally occurring pesticides. Science 226: 184-187.
Nehlig A, Daval JL, Debry G, 1992. Caffeine and the central nervous system: mechanisms of action, biochemical, metabolic and psychostimulant effects. Brain Res Brain Res Rev 17: 139-170.
Newton R, Broughton LJ, Lind MJ, Morrison PJ, Rogers HJ, Bradbrook ID, 1981. Plasma and salivary pharmacokinetics of caffeine in man. Eur J Clin Pharmacol 21: 45-52.
O'Connell SE, Zurzola FJ, 1984. Rapid quantitative liquid chromatographic determination of caffeine levels in plasma after oral dosing. J Pharm Sci 73: 1009-1011.
Okano J, Nagahara T, Matsumoto K, Murawaki Y, 2008. Caffeine inhibits the proliferation of liver cancer cells and activates the MEK/ERK/EGFR signalling pathway. Basic Clin Pharmacol Toxicol 102: 543-551.
Ortweiler W, Simon HU, Splinter FK, Peiker G, Siegert C, Traeger A, 1985. [Determination of caffeine and metamizole elimination in pregnancy and after delivery as an in vivo method for characterization of various cytochrome p-450 dependent biotransformation reactions]. Biomed Biochim Acta 44: 1189-1199.
Schmidt B, Roberts RS, Davis P, Doyle LW, Barrington KJ, Ohlsson A, Solimano A, Tin W, 2006. Caffeine therapy for apnea of prematurity. N Engl J Med 354: 2112-2121.
Serini G, Bussolino F, 2004. Common cues in vascular and axon guidance. Physiology (Bethesda) 19: 348-354.
Uefuji H, Ogita S, Yamaguchi Y, Koizumi N, Sano H, 2003. Molecular cloning and functional characterization of three distinct N-methyltransferases involved in the caffeine biosynthetic pathway in coffee plants. Plant Physiol 132: 372-380.
Yu HH, Houart C, Moens CB, 2004. Cloning and embryonic expression of zebrafish neuropilin genes. Gene Expr Patterns 4: 371-378.