指導教授：陳秀男臺灣大學：漁業科學研究所梁栩福Leong, Eddie Shu PuEddie Shu PuLeong2014-11-302018-07-062014-11-302018-07-062014http://ntur.lib.ntu.edu.tw//handle/246246/263813本研究主要探討菇蕈多醣體（mushroom β-glucan）對體外培養之南美白蝦血淋巴細胞之影響。本實驗將南美白蝦血淋巴細胞進行初代培養（primary culture），結果發現調整滲透壓之培養基組別顯著提高了南美白蝦血淋巴細胞活性並延長存活時間。培養基添加多醣體之後發現，0.5%濃度組別可以延長血淋巴細胞活性維持。將滲透壓調整為900±5 mOsm/Kg 的培養基中添加多醣體，發現可以降低南美白蝦血淋巴細胞凋亡比例；細胞中的ROS產生率會隨著多醣體濃度提升，會有下降再上升的情形；細胞吞噬作用有顯著性的提升，且細胞大小與顆粒性會有複雜化的現象。結果也發現，血淋巴細胞於體外培養時會有類似黑化作用及胞封作用的情形發生，並且隨著多醣體刺激濃度的升高，三種細胞的顆粒性與大小以流式細胞儀監測觀察下有趨向同一種型態的趨勢。The present study aims to examine the effects of mushroom β-glucan on the cultured Litopenaeus vannamei haemocytes via in-vitro experiment. The cell viability and activity were enhanced, while the osmolarity of cell cultured medium was adjusted into the 900±5mOsm/Kg compared to the unadjusted （315±5mOsm/Kg） cultured medium. Further examination also presented that the adjusted cell cultured medium additional supplemented with the 0.5% β-glucan induce the prolonger of the cell survival compared to the unadjusted group. In the ROS production and apoptosis examination, while the shrimp haemocytes cultured in the adjusted cell cultured medium supplemented with the β-glucan presented that apoptosis cituation of the haemocyte was to be reduced but the ROS production was to be enhanced. The phagocytosis ability, cell particles and sizes of the haemocyte cultured in the adjusted cell culture medium supplemented with β-glucan, we observed that the complication of the intracellular particle and the cell size were to be complicated via the FLOW Cytometry observation and the phagocytosis ablity was to be increased. In this research, we observed that the osmolarity of the shrimp cultured medium was supposed to be adjusted in to 900 ±5mOsm/Kg to maintain the cell viability and activity compared to the unadjusted culture medium in the lab experiment. And we also presented that the β-glucan exactly induce the survival rate, phagocytosis activity and the ROS production of shrimp haemocytes, but the reduce the apoptosis of the shrimp haemocytes via the in-vitro experiment.第一章 前言 ...........................................1 第二章 文獻整理.........................................3 第一節 南美白蝦.........................................3 第二節 甲殼類免疫機制....................................6 第三節 初代細胞培養系統..................................14 第四節 流式細胞儀原理介紹................................18 第五節 免疫激活物.......................................20 第六節 研究動機與目的....................................23 第三章 材料與方法.......................................24 第一節 實驗材料.........................................24 第二節 實驗設計.........................................27 第三節 實驗方法.........................................29 第四節統計分析..........................................32 第四章 結果............................................33 第一節 滲透壓改變對南美白蝦血淋巴細胞活性之影響............33 第二節 菇蕈多醣體對南美白蝦血淋巴細胞活性之影響............34 第三節 菇蕈多醣體對南美白蝦血淋巴細胞凋亡之影響............35 第四節 菇蕈多醣體對南美白蝦血淋巴細胞ROS產生率之影響.......36 第五節 菇蕈多醣體對南美白蝦血淋巴細胞吞噬作用之影響....37 第六節 三種南美白蝦血淋巴細胞分選及確認..................38 第五章 討論........................................39 第六章 結論.........................................48 參考文獻............................................49 圖表附件............................................735697926 bytesapplication/pdf論文公開時間：2014/08/21論文使用權限：同意有償授權(權利金給回饋本人)南美白蝦血淋巴細胞細胞培養滲透壓流式細胞儀菇蕈多醣體thesishttp://ntur.lib.ntu.edu.tw/bitstream/246246/263813/1/ntu-103-R01b45025-1.pdf