臺灣博碩士論文加值系統

甲殼類的血球細胞在免疫系統上具有不同的免疫功能。本實驗的目的在探討甲殼類動物螯蝦的血球細胞是否具有一氧化氮合成酶 (Nitric oxide synthase，NOS) 活性以及一氧化氮 (Nitric oxide，NO) 參與之免疫反應。克氏螯蝦 (Procambarus clarkii) 與一般甲殼類相同具三種類型血球細胞：透明細胞、半顆粒細胞和顆粒細胞。生化分析顯示，克氏螯蝦血球細胞萃取液中具有 Ca2+ - independent NOS。體外實驗發現，血球細胞與細菌共同培養時，細菌有附著 (adhesion) 於血球細胞之現象。外加sodium nitroprusside (SNP，NO 釋放物) 於血球細胞與細菌之共同培養時，有細菌附著的完整與破裂血球細胞數有增加之趨勢。當外加內毒素 Lipopolysaccharide (LPS，NOS 誘導物) 參與反應時，細菌附著的效果在完整與破裂血球細胞數皆有顯著的上升，同時 LPS-誘發之細菌附著可被 NG-monomethyl-L-arginine (L-NMMA，NOS抑制劑) 所阻斷。此外，在 SNP 或 LPS 處理下，有細菌附著的完整血球細胞數比破裂血球細胞較早發生。最後，平板測試證實外加 LPS 於血球細胞與細菌共同培養時，細菌的菌落數顯著降低，且 L-NMMA 可阻斷 LPS-誘發之下降菌落數，顯示 LPS 可能誘發 NOS 的表現，導致更高的殺菌活性。綜合上述結果，克氏螯蝦血球細胞中具有誘發性 Ca2+ - independent NOS，其產生之 NO 免疫功能可促使細菌附著於血球細胞並誘發血球細胞破裂的效應，並增加細胞殺菌活性。

In crustaceans, circulating hemocytes assume various immune functions. The present study determined the presence and biochemical characteristics of nitric oxide synthase (NOS) and investigated the immune roles of nitric oxide (NO) in the hemocytes of the crayfish Procambarus clarkii. Morphological analysis identified three types of hemocytes, namely, hyaline, semigranular, and granular cells. Biochemical analysis indicated the presence of a Ca2+-independent NOS in the homogenates of the hemocytes. When bacteria and hemocytes were co-incubated in vitro, adhesion of bacteria around intact and lysed hemocytes was observed. Addition of sodium nitroprusside (SNP; a NO releaser) to the incubation led to a significant increase in the numbers of intact or lysed hemocytes that bacteria adhered to. Similar to SNP, lipopolysaccharide (LPS; a NO inductor) also elicited bacterial adhesion, but this LPS-induced adhesion is prevented by the addition of NG-monomethyl-L-arginine (L-NMMA; a NOS inhibitor). Furthermore, when induced by either SNP or LPS, significant increase in the numbers of bacteria-adhered intact hemocytes occurred prior to that of bacteria-adhered lysed hemocytes, implying bacterial association promotes hemolysis. Finally, standard plate count assay demonstrated that addition of LPS to the hemocyte/bacterium co-incubation resulted in a significant decrease in bacterial colony forming unit (CFU) and that L-NMMA reversed this LPS-induced decrease in bacterial CFU, suggesting that LPS induces NOS expression that ultimately leads to a higher bactericidal activity. In summary, the combined results indicated the presence of Ca2+- independent inducible NOS in the hemocytes of the crayfish Procambarus clarkii. NO, generated by induced NOS, is presumably involved in defending against invading pathogens via promoting bacterial adhesion and hemolysis that subsequently elevate cellular cytotoxicity.

中文摘要……………………………………………….Ⅰ
英文摘要……………………………………………….Ⅱ
圖表目次……………………………………………….Ⅴ
前言…………………………………………………….1
材料與方法…………………………………………….12
結果…………………………………………………….22
討論…………………………………………………….29
參考文獻……………………………………………….37
圖表…………………………………………………….44

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