臺灣博碩士論文加值系統

　 台灣鮑魚 (Haliotis diversicolor)，又名九孔，為本省重要的經濟養殖貝類，在2002~2003年間，台灣東北部養殖場爆發大量死亡疫情，病毒感染被認為是導致此次九孔大量死亡之病原。本實驗的目的在於建立九孔的細胞培養系統，以做為將來病毒感染研究的平台。先導實驗證實，九孔細胞之貼附率會隨著 L-15 以及 Grace 培養基濃度的上升而增加，因此培養基的滲透壓值可能為決定細胞貼附的重要因子。之後以鹽類補充調整的 L-15 培養基實驗中可觀察到九孔卵巢細胞在具有高滲透壓值 (735~1,110 mOsmkg-1)之 L-15培養基中可以順利貼附，而培養於以鹽類補充液調高滲透壓為 838~940 mOsmkg-1 的 L-15 培養基 (LS15, LS18)的九孔卵巢細胞更具有增殖的現象。為了找尋培養九孔細胞之培養基條件，除了對 L-15 和 Grace 培養基進行組合修飾外，也對九孔血淋巴之組成進行分析，並根據分析結果配製出新的培養基-MAH。比較九孔卵巢細胞及血球細胞培養於修飾後的 LS20、GLS15 和 MAH 三種培養基之生長情形，結果顯示 GLS15為培養九孔細胞之最佳培養基，雖然繼代後的細胞無法順利再貼附，却可以繼代培養出懸浮性的細胞，目前培養於 GLS15 培養基之懸浮性卵巢細胞可以成功繼代 8 次，最多存活 85 天；懸浮性血球細胞則成功繼代 11 次，最多存活 96 天。而在培養基添加物實驗中發現，九孔血淋巴萃取物之添加可增加九孔血球細胞的貼附與生長，而胰島素之添加對血球細胞並無明顯之影響。

Taiwan abalone (Haliotis diversicolor) is one of the important economical aquaculture shellfish in Taiwan. In 2002-2003, a massive death break out occurred in north-east of Taiwan, and virus was found to be the causative pathogen. The objective of this study is to establish tissue culture system of Taiwan abalone as the platform for studying virus infection in the future. In the preliminary data, the adhesion rate of Taiwan abalone cells was augmented by rising the concentration of L-15 and Grace’s medium. Therefore, the medium osmolarity is probably the key factor for Taiwan abalone cells adhesion. We also observed that high osmolarity (735~1,110 mOsmkg-1) of L-15 medium can promote abalone ovarian cells adhesion, and the proliferation can also be stimulated by the supplement of salt solution. In order to search better condition of abalone cell culture, we have developed a new medium MAH by modifying the formula of L-15 and Grace’s medium and also from the results of component assay of Taiwan abalone hemolymph extract. Comparing the growth of Taiwan abalone ovarian and hemocyte cells in modified LS20, GLS15 and MAH media, the best medium is GLS15. Although the adhesion cells are unable to attach after subculture, the suspension cells can be passaged. Till now, the suspension ovarian cells can be subcultured for 8 times, alive for 85 days; the suspension hemocyte can be subcultured for 11 times, alive for 96 days. In the medium supplement study, the supplement of abalone hemolymph extract can promote adhesion and proliferation of hemocyte, but not of insulin.

謝誌................................................................................................................I
中文摘要.....................................................................................................III
英文摘要......................................................................................................V
目錄.............................................................................................................VI
表目錄.........................................................................................................IX
圖目錄..........................................................................................................X
第一章 前言.................................................................................................1
一、 台灣鮑魚之簡介..............................................................................1
二、 九孔在臺灣的養殖概況與經濟重要性..........................................2
三、 九孔大量死亡疫情與病毒病害的關係..........................................3
四、 研究動機以及無脊椎動物細胞體外培養之文獻整理..................4
第二章 材料與方法.....................................................................................9
一、 實驗材料..........................................................................................9
二、 實驗方法........................................................................................12
三、 以L-15及 Grace’s 培養基進行九孔細胞的初代培養...............15
(一) 九孔細胞對不同濃度 L-15 培養基之適應性分析.................15
(二) 九孔細胞對鹽類修飾後的 L-15 培養基之適應性分析.........16
(三) 九孔細胞對鹽類修飾後的 Grace’s 培養基之適應性分析....18
四、 九孔血淋巴之組成份分析............................................................19
(一) 有機酸分析................................................................................19
(二) 鹽類離子分析............................................................................20
(三) 游離胺基酸分析........................................................................21
(四) 單醣分析....................................................................................22
五、九孔細胞對於 3 種不同成份培養基的適應性分析之比較…...23
六、九孔血淋巴萃取液及胰島素的添加對九孔血球細胞生長之影響....................................................................................................27
第三章 結果.............................................................................................30
一、 以L-15及 Grace’s培養基進行九孔細胞的初代培養..............30
(一) 九孔細胞對不同濃度 L-15培養基之適應性分析.................30
(二) 九孔細胞對鹽類修飾後的 L-15 培養基之適應性分析........30
(三) 九孔細胞對鹽類修飾後的 Grace’s 培養基之適應性分析....31
二、 九孔血淋巴之組成分析...................................................………32
(一) 有機酸分析................................................................................32
(二) 鹽類離子分析............................................................................32
(三) 游離胺基酸分析........................................................................33
(四) 單醣分析....................................................................................33
三、 九孔細胞對於 3 種不同成份培養基的適應性分析之比較….33
四、 九孔血淋巴萃取液及胰島素的添加對九孔血球細胞生長之影
響...................................................................................................35
第四章 討論.............................................................................................36
第五章 參考文獻.....................................................................................44
第六章 表.................................................................................................49
第七章 圖.................................................................................................57
第八章 附錄.............................................................................................75

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