激發草蝦Penaeus monodon血球細胞產生殺菌物質 活性氧族群之分析

Abstract

以 Nitroblue tetrozolium ( NBT ）染色法定性具有吸附能力的血球細胞所產生的Superoxide anion ( O2-），結果以 Phorbol myristate acetate ( PMA ）處理後有9. 1 ％血球細胞質中產生藍色顆粒， Zymosan 及β-glucan 分別為31％及41.2% ，對照組則為5.1％。利用NBT染色法定量O2-，結果顯示PMA的刺激效果以濃度250ng / ml 最佳，Zymosan 和β－glucan 的濃度則分別為 2 . 5 mg ／ml 和2 mg ／ml最佳。在最佳濃度條件下，比較三種不同免疲刺激物對同一隻蝦血球的激發效果，利用 NBT 定量分析O2- ，發現β-glucan 最佳，可達對照組的 2 . 5 倍；其次為Zymosan ，可達對照組 2 倍；而 PMA 最低，只有 1 . 3 倍。以酚紅氧化法（Oxidation of phenol red）定量血球細胞所產生的 Hydrogen peroxide ( H2O2 ) 結果以β-glucan的刺激效果最佳，為 12.2n mole/mg protein ；其次為Zymosan : 7 . 2 n mole／mg protein和 PMA : 2 .62 n mole/mg protein 。利用Luminol 放大的化學冷光法分析血球接受刺激後是否產生 Hypochlorite ( OCl-），結果發現以 Zymosan 或β-glucan 刺激後，均無法到得冷光；以 PMA 刺激血球，只在血球濃度 5×10^7 cells / ml 、 Luminol 濃度 0 . 1 mM 、 PMA 500 ng／ml及 24℃的條件下測得明顯的化學冷光，但產生之冷光最高亦僅有 1 . 7 mV 。利用 H 2 O2為受質、 guaiacol 做為電子接受者分析草蝦血球打碎後之粗萃取液，發現其中含有類似人頸 Myeloperoxidase ( MPO ）的酵素活性。此類 MPO酵素在 pH7 、0.4 mM H2O2及 2 % NaCl時活性最佳，為 0.104 units/mg protein 。三種刺激物分別激發血球後，測量酵素 MPO 活性，結果僅 PMA 對酵素活性有促進的功能，約為對對照組的 2 .23 倍，而Zymosan 及β-glucan 激發的血球則和對照組血球的MPO 活性沒有差異。上述結果顯示 O2-及H2O2是草蝦血球細胞產生的主要殺菌物質，MPO 所催化形成 OCl-則較次要。三種免疫刺激物中以β-glucan對 O2-和 H2O2的刺激效果表佳，因此建議選取β-glucan 做為免疫剌激物以增加草蝦的非專一性免疫能力.

We used NBT staining to determine the superoxide anion (O2-)produced by the hemocytes which derived from tiger shrimp (Peneaus monodon) and the hemocytes was attached to the cover glass. When cells were treated with PMA ,blue granules were observed in the cytoplasm of 9.1% of the hemocytes. For zymosan-treated,β-glucan-treated and control cells, the percentages of hemocytes showing similar blue granules were 31%,41.2% and 5.1% respectively. Superoxide anions measurements revealed that the best stimulative concentration of PMA、zymosan and β-glucan were 250 ng/ml、2.5mg/ml and 2mg/ml respectively. A comparison of the stimulative effect of these three substances on hemocytes suspensions collected from a single individual tiger shrimp showed that 3-glucan had the strongest effect, followed by zymosan and PHA (2.5,2 and 1.3 times that of the control group, respectively). After oxidizing phenol red to measure the hydrogen peroxide (H202) produced by the observed hemocytesβ-glucan has the strongest stimulative effect (produced 12.2 n mole/mg protein),followed by zymosan and PMA (7.2 and 2.62 n mole/mg protein, respectively). Luminol-enhanced chemilu－minescence analysis of hypochiorites (OCl-) produced by the experimental hemocytes showed that zymosan andβ-glucan had no stimulative effect on OC1- production. Under conditions of 5×10－7 ceils/ml,0.1mM luminol and 500ng/ml PMA at 24℃ .maximum chemiluminescence was only 1.7 mV. Using H2O2 as the substrate and guaiacol as an electron acceptor, we analyzed the enzyme activity of crude extract derived from broken hemocytes; we observed enzyme activity simlar to that of human myeloperoxidase (MPO)(0.104 units/mg protein detected at pH 7 ,0.4 mM H2O2 and 2% NaC1). Our data showed that only PMA had any stimulative effect on MPO－like enzyme activity ( 2.23 times that of the control group); zymosan and β-glucan did not have any observable effect on this activity.