臺灣博碩士論文加值系統

真牡蠣 (Crassostrea gigas)為本省重要的養殖貝類，本論文為了瞭解真牡蠣血球細胞種類與其在防禦機制中所扮演的角色，首先利用光學與電子顯微鏡術觀察真牡蠣的血球型態與細微構造。在倒立位相差顯微鏡下進行未染色之牡蠣血球細胞的活體觀察，依據細胞質中顆粒的有無將真牡蠣的血球細胞區分為顆粒球(granulocyte)和非顆粒球(agranulocyte)兩大類，另有一種數量極少的細胞，暫時將之歸為無法分類之囊泡血球(vesicular hemocytes)。在光學顯微鏡下以100%甲醇和 1%戊二醛溶液兩種固定液，配合Quick-Differ、May-Grünwald及Giemsa三種染色法，除了確認牡蠣血球細胞可分為顆粒球、非顆粒球以及囊泡血球外，更可依顆粒的染色性，再將顆粒球再分為嗜伊紅球、大顆粒嗜鹼球、小顆粒嗜鹼球及兼俱嗜伊紅與嗜鹼性等四種。在穿透式電子顯微鏡的觀察下，顆粒球有少數細長偽足的伸出；粒線體、粗面內質網、還有一些疑似平滑內質網的管狀或圓狀構造散佈細胞質中；最明顯的是胞質內有兩種呈圓形或橢圓狀的顆粒，一種是電子致密，另一種則是電子澄清顆粒。非顆粒球有較多管狀偽足伸出的情形，胞器大多位於核的周邊，而胞質外圍呈現均勻電子緻密的情形；有些非顆粒球中，可發現有殘存小體 (residual body) 的電子緻密顆粒存在。
以流式細胞儀 (flow cytometry) 配合percoll的機度離心分析血球細胞之族群分布，發現牡蠣血球有A、B和C三個族群，各占62.8% 、20.6 %、3.9%，其中A群細胞應為非顆粒球部份，而20.6 %的B群細胞應為顆粒球部份，這和之前在光學顯微鏡下觀察到血球非顆粒與顆粒球的比例相符合(64%：36%)；而數量最少且細胞最大的C群細胞(3.9%)可能是無法分類的囊泡血球。
最後部分進行牡蠣血球的脂質、多醣類以及b-Glucuronidase、a-Naphthyl acteate esterase、Naphthol AS-D chloroacetate esterase、Acid phosphatase、Alkaline phosphate、Peroxidase六種酵素的染色觀察。結果發現脂質染色中，呈陽性反應的皆是顆粒球；而真牡蠣顆粒球與非顆粒球都是PAS陽性反應。而以上六種酵素反應均存在於牡蠣的顆粒球內。利用超氧根氧離子的測定(NBT)和螢光乳珠的吞噬作用，發現非顆粒球與顆粒球皆有吞噬能力，因為吞噬作用後可能會在細胞質中留下肝糖，和以上牡蠣血球皆呈PAS陽性的結果相結合，證實真牡蠣的非顆粒球與顆粒球皆有吞噬能力。

In order to understand the cellular defense system in the oyster, Crassostrea gigas, hemocytes of the Crassostrea gigas were characterized using phase contrast, light and electron microscopy. Three types of hemocytes, which we named granulocyte, agranocyte and vecicular hemocyte, were recognized. Three types of granulocyte were identified by light microscopy, in accordance with the presence of basophilic or eosinophilic granules or a mixture of both in the cytoplasm.
Electron microscopic observations showed that the granulocyte population consisted of two types of cells with ultrastructural similarities but with distinctive granules. One of the two types of granulocytes was characterized by the presence of membrane-bound electron-lucid granules, the second type contained membrane-bound electron-dense granules. The cytoplasm of the granulocyte contained some mitrochondria and endoplasmic reticulum and numerous small membrane bound vesicles. Agranulocyte presented rough endoplasmic reticulum and mitrochondria was often located near the nucleus and some of them contained residual bodies.
With flow cytometry and the discontinuous percoll density gradient centrifugation analyzing, the hemocyte population contains three subpopulations A (62.8%), B (20.6%) and C (3.9%) in oyster. According to the results of these analysis, supposing that subpopulation A is agranulocyte, subpopulation of B is garnulocyte and maybe subpopulation C is vesicular hemocytes.
Results of the histochemistry staining showed that granulocyte was lipid postive and the haemocyte were b-glucuronidase, a-naphthyl acteate esterase, naphthol AS-D chloroacetate esterase, acid phosphatase, alkaline phosphate and peroxidase positive. Moreover, the granulocyte and agranulocyte were PAS postive and showed phagocyte ability against latex and zymosan particles.

目 錄
摘要 ........................................................I
前言 ........................................................1
文獻回顧 ....................................................3
一、二枚貝血球細胞之分類.....................................3
二、二枚貝的血球生成 ........................................4
三、二枚貝血球細胞種類和型態特徵 ............................5
四、二枚貝血球的功能 ........................................8
五、二枚貝血球在防禦機制中扮演的角色 ........................8
一、真牡蠣血球細胞之形態觀察................................ 18
(一) 實驗材料 ...............................................18
(二) 研究方法 ...............................................18
1、血淋巴之抽取以及牡蠣血球玻片之製作....................... 18
2、倒立顯微鏡觀察 ...........................................18
3、光學顯微鏡觀察 ...........................................18
4、牡蠣血球細胞之穿透式電子顯微鏡 (TEM) 觀察 ...............19
二、真牡蠣血球之族群分佈與分離 ..............................20
(一) 實驗材料 ...............................................20
(二) 研究方法 ...............................................20
1、Percoll分離...............................................21
2、以流式細胞儀 (flow cytometry) 分析血球細胞之族群分布......21
三、真牡蠣血球細胞之組織化學染色觀察.........................21
(一) 實驗材料－各種酵素染色試劑的配製........................22
(二) 研究方法................................................23
1、脂質染色..................................................23
2、多醣類染色 (Periodic acid-schiff, PAS)....................24
3、Lysosome 染色.............................................24
4、鹼性磷酸酶 (Alkaline phosphatase) 染色....................24
5、酸性磷酸酶 (Acid phosphatase) 染色解脂酶..................24
6、b-Glucuronidase 染色......................................25
7、解脂酶 (a-Naphthyl acetate esterase) 染色.................25
8、解脂酶 (Naphthol AS-D chloroacetate esterase) 染色........25
9、過氧化酶 (Peroxidase) 染色................................26
10、吞噬作用.................................................26
結果.........................................................27
一、真牡蠣血球細胞之形態觀察.................................27
1、倒立顯微鏡觀察 ...........................................27
2、光學顯微鏡觀察 ...........................................28
3、牡蠣血球細胞之穿透式電子顯微鏡 (TEM) 觀察.................29
二、真牡蠣血球之族群分佈與分離...............................30
1、Percoll分離...............................................30
2、以流式細胞儀 (flow cytometry) 分析血球細胞之族群分布......30
三、真牡蠣血球細胞之組織化學染色觀察.........................31
1、脂質染色..................................................31
2、多醣類染色 (Periodic acid-schiff, PAS)....................31
3、Lysosome 染色............................................ 31
4、鹼性磷酸酶 (Alkaline phosphatase) 染色....................31
5、酸性磷酸酶 (Acid phosphatase)染色 ........................32
6、b-Glucuronidase 染色......................................32
7、解脂酶 (a-Naphthyl acetate esterase) 染色.................32
8、解脂酶 (Naphthol AS-D chloroacetate esterase) 染色........33
9、過氧化酶 (Peroxidase) 染色................................33
10、吞噬作用.................................................33
討論.........................................................34
參考文獻.....................................................43
圖表.........................................................57

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