由於共生渦鞭毛藻與珊瑚蟲之間的胞內共生，使得珊瑚礁可以建立在營養貧乏的熱帶海域，並提供海洋生物生長多樣的棲地。然而我們對此胞內共生的細胞分子機制，至今仍了解有限，其瓶頸在於無法建立一個遺傳上可以操作的系統，以驗證胞內共生相關基因所扮演的功能。本研究要旨在發展一個可在腔腸動物上操作的基因轉染技術，將目標基因轉染到刺胞動物細胞核內並表現。實驗材料採用美麗海葵作為模式動物，選殖到美麗海葵的histone基因，並將之送入pEGFP-C1表現載體中，利用CPPs將pEGFP-Ap表現質體送入COS7細胞中表現，其綠色螢光融合蛋白的表現位置也位於細胞核內。在腔腸動物美麗海葵模式物種中，實驗結果顯示CPPs-FITC可以進入美麗海葵細胞中與細胞核內，並且能取代Hoechst33258與DNA的結合。更進一步的分析更發現，CPPs的確能攜帶Cy3標記的引子DNA進入美麗海葵細胞核內，強烈暗示CPPs作為細胞轉染試劑（如lipofectamine）的潛力。總結來看，所有結果都支持CPPs適用於海洋腔腸動物轉染的試劑之一，但因效率尚未達到最佳，如何調整條件達到轉染效率最佳化是未來研究重點。

Intracellular symbiosis between Symbiodinium dinoflagellates and corals forms coral reefs providing diverse and essential habitats for the existence of a large variety of marine organisms possible in the nutrient-poor tropical seas. However, our knowledge on the cellular and molecular mechanisms of this important ecological interaction is poor, partly due to a lack of a genetically manipulatable model of cnidarian. The aim of the present study is to establish a transfection system in a cnidarian model, Aiptasia pulchella, with cell-penetrating peptides (CPPs) as the delivery vehicle. I have successfully established a CPPs-based protocol to deliver pEGFP-ApHistone reporter plasmid into COS7 cells and expressed EGFP-ApHistone fusion protein in the nucleus. The results showed that the CPPs were able to penetrate A. pulchella cells and replaced the binding between DNA and Hoechst33258, as CPPs were not only penetrated into cells but also diffused into nucleus in the marine cnidarian cell model. Moreover, the results strongly suggests that CPPs carried the Cy3-conjugated DNA primers into the nucleus of A. pulcehlla endodermal cells, revealing its potentials for cell transfection reagents. Overall, all results provided evidence that the CPPs are one of transfection candidates for marine cnidarian even the efficiency was low under current protocol. Optimization of proceduces is underway to improve the delivery efficiency.

論文審定書....................................................................................................................ⅰ
中文摘要........................................................................................................................ⅱ
英文摘要........................................................................................................................ⅲ
目錄……………………………………………………………………………………ⅳ
圖目錄…………………………………………………………………………………ⅵ
第一章　緒論…………………………………………………………………………..1
第一節　背景與動機..............................................................................................1
第二節　研究目的與重要性..................................................................................7
第二章　材料與方法......................................................................................................9
第一節　實驗動物與細胞………………………………………………………..9
第二節　COS7細胞繼代方式…………………………………………………...9
第三節　RNA抽取與cDNA基因庫製作………………………………………9
第四節　ApHistone的基因選殖序列分析……………………………………..10
第五節　轉形……………………………………………………………………11
第六節　質體DNA抽取………………………………………………………..11
第七節　DNA膠體電泳…………………………………..…............................11
第八節　CPPs導引的細胞轉染與EGFP reporter分析……………………….11
第九節　CPPs與CPPs-FITC溶液製備……………………………………….12
第三章　結果、討論與建議........................................................................................13
第一節　結果與討論……………………………………………………………..13
第二節　建議……………………………………………………………………..16
第四章　結論…………………………………………………………………………17
參考文獻………………………………………………………………………………19

[1] Stanley GD Jr, Photosymbiosis and the evolution of modern coral reefs, Science, 312 (2006) 857-858.
[2] Baker AC, Flexibility and specificity in coral-algal symbiosis: diversity, ecology, and biogeography of symbiodinium, Annual Review of Ecology, Evolution, and Systematics, 34 (2003) 661-689.
[3] Volkov I, Banavar JR, Hubbell SP, Maritan A , Patterns of relative species abundance in rainforests and coral reefs, Nature, 450 (2007) 45-49.
[4] Rowan R, Coral bleaching: thermal adaptation in reef coral symbionts, Nature, 430 (2004) 742-742.
[5] Cantin NE, van Oppen MJH, Willis BL, Mieog JC, Negri AP, Juvenile corals can acquire more carbon from high-performance algal symbionts, Coral Reefs, 28 (2009) 405-414.
[6] Muscatine L, Falkowski PG, Porter JW, Dubinsky Z, Fate of Photosynthetic fixed carbon in light- and shade-adapted colonies of the symbiotic coral stylophora pistillata, Proceedings of the Royal Society of London B: Biological Sciences, 222 (1984) 181-202.
[7] Jacques TG, Pilson MEQ, Experimental ecology of the temperate scleractinian coral Astrangia danae I. Partition of respiration, photosynthesis and calcification between host and symbionts, Marine Biology, 60 (1980) 167-178.
[8] Richardson DHS, Douglas A., The symbiotic habit, Symbiosis, 51 (2010) 197-198.
[9] Janeway CA Jr, Medzhitov R. , Innate immune recognition, Annual Review of Immunology, 20 (2002) 197-216.
[10] McGuinness DH, Dehal PK, Pleass RJ, Pattern recognition molecules and innate immunity to parasites, Trends in Parasitology, 19 (2003) 312-319.
[11] Underhill DM, Ozinsky A, Phagocytosis of microbes: complexity in action, Annual Review of Immunology, 20 (2002) 825-852.
[12] Markell DA, Trench RK, Iglesias-Prieto R., Macromolecules associated with the cell walls of symbiotic dinoflagellates, Symbiosis, 12 (1992) 19-31.
[13] Markell DA, Trench RK, Macromolecules exuded by symbiotic dinoflagellates in culture: amino acid and sugar composition1, Journal of Phycology, 29 (1993) 64-68.
[14] Markell D, Wood-Charlson E, Immunocytochemical evidence that symbiotic algae secrete potential recognition signal molecules in hospite, Marine Biology, 157 (2010) 1105-1111.
[15] Meints RH, Pardy RL, Quantitative demonstration of cell surface involvement in a plant-animal symbiosis: lectin inhibition of reassociation, Journal of Cell Science, 43 (1980) 239-251.
[16] Rutishauser U, Sachs L, Cell-to-cell binding induced by different lectins, The Journal of Cell Biology, 65 (1975) 247-257.
[17] Lin KL, Wang JT, Fang LS, Participation of glycoproteins on zooxanthellal cell walls in the establishment of a symbiotic relationship with the sea anemone, Aiptasia pulchella, Zoological Studies, 39 (2000) 172-178.
[18] Wood-Charlson EM, Hollingsworth LL, Krupp DA, Weis VM, Lectin/glycan interactions play a role in recognition in a coral/dinoflagellate symbiosis, Cellular Microbiology, 8 (2006) 1985-1993.
[19] Bay LK, Cumbo VR, Abrego D, Kool JT, Ainsworth TD, Willis BL , Infection dynamics vary between symbiodinium types and cell surface treatments during establishment of endosymbiosis with coral larvae, Diversity, 3 (2011) 356-374.
[20] Colley N J, Trench RK, Selectivity in phagocytosis and persistence of symbiotic algae in the scyphistoma stage of the jellyfish Cassiopeia xamachana, Proceedings of the Royal Society of London. Series B, Royal Society (Great Britain), 219 (1983) 61-82.
[21] Schwarz JA, Krupp DA, Weis VM, Late larval development and onset of symbiosis in the Scleractinian coral Fungia scutaria, The Biological Bulletin, 196 (1999) 70-79.
[22] Rodriguez-Lanetty M, Wood-Charlson EM, Hollingsworth L , Krupp D , Weis VM, Temporal and spatial infection dynamics indicate recognition events in the early hours of a dinoflagellate/coral symbiosis, Marine Biology, 149 (2006) 713-719.
[23] Wilkerson FP, Muscatine L, Uptake and Assimilation of Dissolved Inorganic nitrogen by a symbiotic sea anemone, Proceedings of the Royal Society of London B: Biological Sciences, 221 (1984) 71-86.
[24] Hohman TC, McNeil PL, Muscatine L, Phagosome-lysosome fusion inhibited by algal symbionts of Hydra viridis, Journal of Cell Biology, 94 (1982) 56-63.
[25] Chen MC, Cheng YM, Sung PJ, Kuo CE, Fang LS, Molecular identification of Rab7 (ApRab7) in Aiptasia pulchella and its exclusion from phagosomes harboring zooxanthellae, Biochemical and Biophysical Research communications, 308 (2003) 586-595.
[26] Chen MC, Cheng YM, Hong MC, Fang LS, Molecular cloning of Rab5 (ApRab5) in Aiptasia pulchella and its retention in phagosomes harboring live zooxanthellae, Biochemical and Biophysical Research Communications, 324 (2004) 1024-1033.
[27] Chen MC, Hong MC, Huang YS, Liu MC, Cheng YM, Fang LS, ApRab11, a cnidarian homologue of the recycling regulatory protein Rab11, is involved in the establishment and maintenance of the Aiptasia-Symbiodinium endosymbiosis, Biochemical and Biophysical Research Communications, 338 (2005) 1607-1616.
[28] Hong MC, Huang YS, Lin WW, Fang LS, Chen MC, ApRab3, a biosynthetic Rab protein, accumulates on the maturing phagosomes and symbiosomes in the tropical sea anemone, Aiptasia pulchella, Comparative Biochemistry and Physiology. Part B, Biochemistry & Molecular Biology, 152 (2009) 249-259.
[29] Hong MC, Huang YS, Song PC, Lin WW, Fang LS, Chen MC, Cloning and characterization of ApRab4, a recycling Rab protein of Aiptasia pulchella, and its implication in the symbiosome biogenesis, Marine Biotechnology , 11 (2009) 771-785.
[30] Rink J, Ghigo E, Kalaidzidis Y, Zerial M, Rab conversion as a mechanism of progression from early to late endosomes, Cell, 122 (2005) 735-749.
[31] Grosshans BL, Ortiz D, Novick P, Rabs and their effectors: achieving specificity in membrane traffic, Proceedings of the National Academy of Sciences of the United States of America, 103 (2006) 11821-11827.
[32] Kinchen JM, Ravichandran KS, Phagosome maturation: going through the acid test, Nature reviews. Molecular Cell Biology, 9 (2008) 781-795.
[33] Karakashian MW, Karakashian SJ, Intracellular digestion and symbiosis in Paramecium bursaria, Experimental Cell Research, 81 (1973) 111-119.
[34] Karakashian SJ, Rudzinska MA, Inhibition of lysosomal fusion with symbiont-containing vacuoles in Paramecium bursaria, Experimental Cell Research, 131 (1981) 387-393.
[35] Kodama Y, Fujishima M, Secondary symbiosis between Paramecium and Chlorella cells, International Review of Cell and Molecular Biology, 279 (2010) 33-77.
[36] O'Brien TL, Inhibition of vacuolar membrane fusion by intracellular symbiotic algae in Hydra viridis (Florida strain), Journal of Experimental Zoology, 223 (1982) 211-218.
[37] Fitt WK, Trench RK, Endocytosis of the symbiotic dinoflagellate Symbiodinium microadriaticum Freudenthal by endodermal cells of the scyphistomae of Cassiopeia xamachana and resistance of the algae to host digestion, Journal of Cell Science, 64 (1983) 195-212.
[38] Venn AA, Tambutté E, Lotto S, Zoccola D, Allemand D, Tambutté S, Imaging intracellular pH in a reef coral and symbiotic anemone, Proceedings of the National Academy of Sciences, 106 (2009) 16574-16579.
[39] Böttger A, Alexandrova O, Cikala M, Schade M, Herold M, David C, GFP expression in hydra: lessons from the particle gun, Development Genes and Evolution, 212 (2002) 302-305.
[40] Watanabe H, Schmidt HA, Kuhn A, Hoger SK, Kocagoz Y, Laumann-Lipp N, Ozbek S, Holstein TW, Nodal signalling determines biradial asymmetry in hydra, Nature, 515 (2014) 112-115.
[41] Chang M, Chou JC, Lee HJ, Cellular internalization of fluorescent proteins via arginine-rich intracellular delivery peptide in plant cells, Plant & Cell Physiology, 46 (2005) 482-488.
[42] Liu BR, Lin MD, Chiang HJ, Lee HJ, Arginine-rich cell-penetrating peptides deliver gene into living human cells, Gene, 505 (2012) 37-45.
[43] Liu BR, Liou JS, Chen YJ, Huang YW, Lee HJ, Delivery of nucleic acids, proteins, and nanoparticles by arginine-rich cell-penetrating peptides in rotifers, Marine Biotechnology, 15 (2013) 584-595.
[44] Dai YH, Liu BR, Chiang HJ, Lee HJ, Gene transport and expression by arginine-rich cell-penetrating peptides in paramecium, Gene, 489 (2011) 89-97.