臺灣博碩士論文加值系統

九孔 (Haliotis diversicolor) 又稱為台灣鮑魚，為本省重要的養殖經濟貝類。本論文擬探討九孔血球型態、血球相關免疫反應，以及鋅、鎘重金屬對於九孔血球細胞之影響。首先利用光學與電子顯微鏡術觀察九孔血球型態與細微構造，依細胞質中顆粒的有無，將九孔血球分為顆粒球 (granulocyte) 和透明球 (hyalinocyte) 兩大類，顆粒球中的顆粒為嗜伊紅性。在穿透式電子顯微鏡術的觀察下，透明球有偽足伸出，細胞呈圓形或橢圓形、而核通常偏向一邊，細胞質內有粒線體、高基氏體、內質網、殘存小體和液泡等胞器。而顆粒球極少有偽足的伸出，其最明顯的特徵為細胞質內的電子緻密顆粒，顆粒的形狀呈圓形或是橢圓形；細胞中心有圓形、橢圓形或是馬蹄形的核，此外因為顆粒的緣故，所以可觀察到的胞器較少，包括粒線體和高基氏體。嘗試以Percoll分離血球細胞，並無法將血球細胞分離，所以進ㄧ步利用血球細胞本身附著能力不同之特性來分離透明球和顆粒球，結果顯示附著性佳的是透明球，而附著性差的血球細胞為顆粒球。
進行九孔血球細胞的化學和酵素組成之染色觀察，結果九孔血球細胞不帶有脂質，而兩種血球都呈現多醣類的PAS陽性反應。五種酵素染色結果中有四種在透明球中呈現陽性反應，包括酸性磷酸酶、β-葡糖苷酸酶、過氧化酶和氯化醋酸AS-D 萘酚酯酶。評估九孔血球細胞的免疫能力，証實透明球具有吞噬的能力。另外觀察到透明球中超氧根離子產生的情形。此外，並沒有觀察到顆粒球具有吞噬反應。
最後以氯化鋅、氯化鎘海水溶液浸泡處理九孔，藉由呼吸爆(respiratory burst)、超氧歧化酶 ((superoxide dismutase, SOD) 與酚氧化酶(phenoloxidase, PO) 活性的變化，探討重金屬對九孔血球免疫相關反應之影響。針對九孔透明球進行的呼吸爆活性分析結果顯示，重金屬鋅和鎘對九孔呼吸爆活性並沒有顯著性的影響。但是同樣針對透明球的PO活性分析中，各組中只有低濃度鋅0.5 ppm和0.05 ppm的處理組，第1、3、7天實驗組的PO活性逐漸上升，在第7天時明顯高於對照組，達到最高值。而收集九孔所有血球細胞所進行之超氧岐化酶的分析結果，重金屬處理結果也沒有顯著性的影響，唯各組數據平均值的標準差值偏高，顯示個體間的差異性大。

Small abalone (Haliotis diversicolor) is an economical aquacultural species in Taiwan. This study focused on three point of hemocyte of abalone, including the morphology of hemocyte, relative immune response and the effect of heavy metal (zinc and Cadmium) treatment. First, we use the light and electronic microscopies for observing the morphology and ultrastructure. Based on the cytoplasmic granules, the hemocytes of abalone were separated into granulocytes and hyalinocytes. The eosinophilc granules were observed in granulocytes. Ultrastructurally, the hyalinocyte was characterized by the presence of several pseudopodia and with various organelles, mitochondria, Golgi apparatus, endoplasmic reticula and residual bodies. The pseudopodia didn’t appear in granulocytes. They were characterized by the presence of numerous cytoplasmic electron-dense granules. The granulocytes were round or oval in shape, most of them consisted of round or U-shaped nucleus. Because of the granules, the organelles observed were fewer than those of hyalinocytes. The hemocytes isolation was not achieved by percoll density gradient centrifugation. However, the adhesion ability of hemocytes could separate the granulocytes from the adherent hyalinocytes.
Using cytochemical staining method, the hemocytes of small abalone were lipid negative reaction, but all the PAS positive reactions. The hyalinocytes showed activity of acid phosphatase, β-glucuronidase, and naphthol AS-D chloroacetate esterase. Most hemocytes proved positive for peroxidase.
Small abalones were exposed to the heavy metal, zinc chloride and cadmium chloride, and examined by respiratory burst, superoxide dismutase (SOD) and phenoloxidase (PO) activities for investigating the heavy metal (Zinc and cadmium) effect on the immuno response of small abalone. There’s no significant respiratory burst response of hyalinocytes of small abalone in zinc and cadmium stress. There’re only low concentrations as 0.5ppm and 0.05ppm in zinc showed the increasing of PO activities at day 1, day 3 and day 7 post exposure. The value was distinct higher than control at day 7. There’re no significant effect of zinc and cadmium stress on the superoxide dismutase (SOD) activities of hemocytes of small abalone. In addition, the standard error of mean value was too high compared to control group, demonstrate the individual variation was big.

頁次
謝辭………………………………………………………………….. i
中文摘要…………………………………………………………….. ii
英文摘要…………………………………………………………….. iv
目錄………………………………………………………………….. vi
表目錄……………………………………………………………….. viii
圖目錄……………………………………………………………….. ix
前言………………………………………………………………….. 1
文獻回顧…………………………………………………………….. 3
材料與方法 ………………………………………………………… 13
一、九孔血球細胞型態之觀察………………………………….. 13
(一) 實驗材料……………………………………………….. 13
(二) 研究方法……………………………………………….. 13
二、九孔血球細胞之族群分離…………………………………… 15
(一) 實驗材料……………………………………………….. 15
(二) 研究方法……………………………………………….. 16
三、九孔血球之細胞化學和酵素染色觀察……………………… 17
(一) 各種染色試劑之配置………………………………….. 17
(二) 染色方法……………………………………………….. 18
四、九孔血球細胞相關之免疫反應評估……………………….. 20
五、重金屬對九孔血球免疫之影響…………………………….. 21
(一) 重金屬溶液之配製…………………………………….. 21
(二) 研究方法……………………………………………….. 21
(三) 統計分析……………………………………………….. 23
結果 24
一、九孔血球細胞型態之觀察………………………………….. 24
二、九孔血球細胞之族群分離…………………………………... 25
三、九孔血球之細胞化學和酵素染色觀察……………………... 26
四、九孔血球細胞相關之免疫反應評估………………………... 27
五、重金屬對九孔血球免疫之影響……………………………... 28
討論 ………………………………………………………………… 30
參考文獻…………………………………………………………….. 37
圖表………………………………………………………………….. 49

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