在現今水產養殖中，大多使用集約式或循環養殖系統，氨是水生環境裡常見的有毒物質，其主要來源包括水生動物的代謝排放與動植物的有機質分解所產生，而在高密度的養殖環境中容易因生物代謝排放與飼料殘餌，造成氨在短時間內大量累積。容易使得生物無法維持生理恆定，最終導致死亡。本研究以全球性重要經濟養殖物種-南美白對蝦(Litopenaeus vanname)作為實驗對象，探討南美白對蝦暴露於亞致死氨濃度(1.5 and 3 mg/L TAN)96小時後，其鰓與血淋巴細胞上蛋白質品質恆定、抗氧化以及細胞凋亡等分子細胞防衛機制的探討。首先在蛋白質恆定機轉中，研究結果指出Heat shock factor 1(HSF1)與Heat shock protein (HSP70)在鰓與血淋巴細胞的mRNA表現量相較於控制組有顯著的增加，失去活性而聚集的蛋白質實驗結果中，各組別沒有顯著差異。在酵素調節的抗氧化系統中，其分子層次上的反應顯示，鰓上的錳超氧化歧化酶(Manganese Superoxide Dismutase，簡稱Mn SOD)、穀胱甘肽過氧化酶(glutathione peroxidase，簡稱GPx)、過氧化氫酶（catalase，簡稱CAT）的mRNA表現量於3 mg/L的暴露下有明顯上升的趨勢，而在總抗氧化能力中，沒有顯著的差異。在血淋巴細胞的實驗結果中，Mn SOD的mRNA表現量相較於處理組，有下降的趨勢，GPx與CAT的mRNA表現量與鰓組織有相同的趨勢，於3 mg/L的暴露下有明顯的上升表現，而在血淋巴細胞的總抗氧化能力中，沒有顯著差異。此外，在細胞凋亡方面，鰓上3種Caspase-3，-4和-5mRNA的表現量於3 mg/L的暴露下明顯增加，在Caspase-2則無顯著差異。在DNA片斷化的實驗結果中也指出在氨暴露下確實對於白蝦的鰓造成片斷化的情形發生，然而在血淋巴細胞的實驗中，4種Caspase-2, -3，-4和-5mRNA的表現量於3 mg/L的氨暴露下明顯增加的趨勢。在DNA片斷化的實驗結果中則與控制組無顯著差異。本篇的實驗結果證實當南美白對蝦面臨亞致死濃度氨暴露下，對南美白對蝦其鰓上細胞以及血淋巴細胞會造成逆境刺激，影響細胞中蛋白質品質之恆定，同時亦可能誘發氧化壓力，因而活化體內的蛋白質品質恆定機轉、抗氧化酶以及細胞凋亡等的分子細胞防衛機制，以抵抗亞致死氨逆境。本次研究將提供相關理學基礎，以更加了解亞致死氨濃度的潛在威脅，以於未來可提供更多科學參考，幫助於預測亞致死濃度氨的毒性和環境監測的研究上。

In present aquaculture, the intensive and recirculating aquaculture systems are universally used. Ammonia is a common toxic substance in aquatic environments, which is excreted by aquatic animals as a by-product of protein catabolism and from the decomposition of feeds and organic matter. Therefore, it can easily rise to high levels to harm to aquatic animals in short-term time. Moreover, excessive ammonia would cause the animals can't maintain physiological homeostasis to eventually lead to death. Pacific white shrimp (Litopenaeus vannamei) is an important worldwide aquaculture species. However, study on the mechanisms of molecular and cellular stress responses employed for coping with sublethal ammonia stress remains limited. In this study, regulatory responses of protein quality control (PQC), antioxidative mechanisms and apoptosis were investigated in shrimp gills after 96h exposure to sublethal ammonia levels (1.5 and 3 mg/L TAN). In PQC mechanism, the study concluded that Heat shock factor 1 (HSF1) and Heat shock protein (HSP70) the mRNA expression in gills and hemocytes compared to the control group had significantly increased. In addition, protein aggregation results in each category no significant differences. Moreover, the transcript levels of antioxidative enzymes, manganese superoxide dismutase (Mn SOD), glutathione peroxidase (GPx) and catalase in gills evidently upregulated at 3 mg/L TAN exposure. In the results of hemocytes, the amount of Mn SOD mRNA expression compared to the treatment group, there was a downward trend, GPx and CAT mRNA expression of the amount of gill tissue has the same tendency, under exposure to 3 mg / L are significant increase. However, in gills and hemocytes no significant difference was found in total antioxidant capacity among all groups, possibly implying the antioxidative enzymes acted a predominant role in antioxidant cytoprotection in white shrimp under ammonia stress. In addition, the results from apoptotic responses revealed that transcript levels of caspase-3, -4 and -5 increased in gills as well as extensive DNA fragmentation was induced obviously in shrimp gills exposed to 3 mg/L TAN. However, in hemocytes experiment, four genes of Caspase-2, -3, -4 and -5mRNA in the 3 mg / L of ammonia exposure under rising trend. The control group with no significant differences in the DNA fragment of the experimental results. This study improved the understandings of vital cytoprotective roles of PQC, antioxidative defenece and apoptosis in coping with sublethal ammonia stress in gills and hemocytes of white shrimp. These findings were suggested to be helpful in predicting sublethal ammonia toxicity and potentially useful in environmental monitoring studies.

論文審定書
論文公開授權書
致謝 i
摘要 ii
Abstract iv
1. Introduction 1
2. Materials and methods 5
2.1. Experimental shrimp and environments 5
2.2. Ammonia exposure 6
2.3. Sample collections 6
2.4. Total RNA extraction and reverse transcription 7
2.5. Quantitative real-time PCR 7
2.6. Cell protein fractionation and isolation of aggregated proteins 9
2.7. Total antioxidant capacity assay 10
2.8. Analysis of DNA fragmentation 10
2.9. Statistical analysis 11
3. Results 12
3.1 Protein quality control mechanism. 12
3.1.1 mRNA expression of heat shock factor 1 (HSF1) and heat shock protein 70 (HSP70) in gills and hemocytes 12
3.1.2 The levels of aggregated proteins 12
3.2 Antioxidant defense mechanism 13
3.2.1 Transcriptional responses of antioxidative enzymes, manganese superoxide dismutase (Mn SOD), glutathione peroxidase (GPx) and catalase (CAT) 13
3.2.2 Total antioxidant capacity assay 13
3.3 Apoptosis 14
3.3.1 mRNA expression of caspase-2, caspase -3, caspase -4 and caspase -5 in gills and hemocytes 14
3.3.2 DNA fragmentation 14
4. Discussion 15
4.1. Integration and contribution 15
4.2. Protein quality control 15
4.3. Antioxidant defense mechanism 17
4.4. Apoptosis 18
4.5. Conclusion 20
5. References 21
6. Tables 27
7. Figures 29

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