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標題: | 南美白蝦血球分離及三種血球免疫基因表現 Observation of Immune Gene Expression and Separated Haemocytes of Pacific White Shrimp, Litopenaeus vannamei |
作者: | Chih-Chiu Yang 楊志秋 |
指導教授: | 陳秀男(Shiu-Nan Chen) |
關鍵字: | 白蝦,血球,免疫基因, white shrimp,haemocyte,immune gene, |
出版年 : | 2015 |
學位: | 博士 |
摘要: | 蝦類的血淋巴細胞在免疫系統上扮演了重要的角色,幾乎所有的免疫反應均是由血淋巴細胞所產生。本研究以全球主要養殖蝦種之一的南美白蝦 Litopenaeus vannamei為研究對象,針對其血淋巴細胞分離過程中,所需使用的緩衝溶液進行調整,使其更貼近血淋巴細胞在蝦體內的狀況,並利用流式細胞分選儀進行不同血淋巴細胞族群的分選,再將分選之後不同族群的血淋巴細胞抽取總核糖核酸,以即時定量PCR進行免疫相關基因,包括proPO、LGBP、Peroxinectin、Crustin、Lysozyme、Penaeidin-3a及Transglutaminase 表現量分析。結果顯示:
當南美白蝦飼養在鹽度分別為10‰、20‰及30‰時,棋血淋巴滲透壓為: 507±43.89 mOsm/kg、779±29.96 mOsm/kg及1136.67±98.17 mOsm/kg。根據此結果,將抗凝劑及流式細胞儀所使用之鞘液的滲透壓調整成1200 mOsm/kg進行實驗。 以流式細胞儀分選系統,根據兩種常用流式細胞參數FSC和SSC,血淋巴細胞可分為三個族群。光學顯微鏡底下觀察發現顆粒球(Granular Cell)、半顆粒球(Semigranular Cell)及透明球(Hyaline Cell)三種不同型態的細胞。免疫相關基因表現是利用即時定量PCR分析不同免疫相關基因與b-actin的相對定量。結果顯示:ProPO,LGBP和peroxinectin高度表現在顆粒球,而LGBP,Crustin,Lysozyme和Penaeidin-3a高度表現在半顆粒球中。Transglutaminase則高度表現在透明球。 Haemocyte of Shrimp played an important role in immune system; all of immune response of shrimp was active by haemocyte. In this study, L. vannamei haemocytes were firstly grouped into three different types and the immune related genes expression in grouped haemocytes were estimated. Those buffers include anticoagulant buffer and sheath fluid that was used by flow cytometery sorting system were modified according different environment of shrimp. White shrimp cultured in 10‰, 20‰ and 30% salinity water, osmolality are 507±43.89 mOsm/kg,779±29.96 mOsm/kg and 1136.67±98.17 mOsm/kg. According to those results, modified osmolality of buffers are 1200 mOsm/kg. Diverse haemocytes from Pacific white shrimp Litopenaeus vannamei were spread by flow cytometer sorting system. Using the two commonly flow cytometric parameters FSC and SSC, the haemocytes could be divided into three populations. Microscopy observation of L. vannamei hemocytes in anticoagulant buffer revealed three morphologically distinct cell types designated as granular cell, hyaline cell and semigranular cell. Immune genes, which includes prophenoloxidase (proPO), lipopolysaccharide--glucan binding protein (LGBP), peroxinectin, crustin, lysozyme, penaeid-3a and transglutaminase (TGase), expressed from different haemocyte were analysed by quantitative real time PCR (qPCR). Results from the mRNA expression were estimated by relative level of each gene to -actin gene. Finally, the seven genes could be grouped by their dominant expression sites. ProPO, LGBP and peroxinectin were highly expressed in granular cells, while LGBP, crustin, lysozyme and P-3a were highly expressed in semigranular cells and TGase was highly expressed in hyaline cells. In this study, L. vannamei haemocytes were firstly grouped into three different types and the immune related genes expression in grouped haemocytes were estimated. |
URI: | http://tdr.lib.ntu.edu.tw/jspui/handle/123456789/52277 |
全文授權: | 有償授權 |
顯示於系所單位: | 漁業科學研究所 |
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