通常遭到眼鏡蛇咬傷的病患在臨床上會表現出系統性神經症狀，例如肌肉無力或呼吸衰竭，還會有局部組織的損傷，例如組織潰爛或嚴重的壞死性筋膜炎。有趣的是糟糕非洲眼鏡蛇N. nivea咬傷的病患，主要表現出系統性神經症狀，沒有明顯的組織損傷，而遭到亞洲眼鏡蛇N. atra 與N. kaothia咬傷的病患，卻沒有明顯的系統性神經症狀，反而有顯著的組織損傷。為了瞭解造成這樣特別影響的可能機制，本實驗室針對N. atra、N. kaouthia和N. nivea的蛇毒進行蛋白質體學的差異性分析研究，並且探討其差異性與細胞毒性的關連性。
根據液相層析、膠體電泳以及質譜分析等方法，可以確認N. atra、N. kaouthia和N. nivea的蛇毒成分主要是由神經毒素(neurotoxins)、心臟毒素(cardiotoxins or cytotoxins)以及磷脂酶A2 (phospholipase A2)所組成，表現量各有不同。相較於兩個在臨床上表現出嚴重組織壞死的亞洲眼鏡蛇，非洲眼鏡蛇N. nivea毒液中磷脂酶A2的含量相對很少，因此我們假設磷脂酶A2可能透過造成細胞死亡，進一步增加局部組織壞死的程度。
我們透過細胞株HEK-293進行細胞毒性測試，來探討磷脂酶A2是否與組織壞死有相關性。研究的結果顯示N. atra、N. kaouthia和N. nivea毒液的IC50分別為~12.2,13.5和17.4μg/ ml。 進一步分析可以發現N. atra、N. kaouthia和N. nivea毒液都可以誘導細胞凋亡(apoptosis)和細胞壞死(necrosis)，而細胞凋亡(apoptosis)是主要的途徑。 從結果來看，我們可以發現磷脂酶A2可能實際參與了由眼鏡蛇咬傷所引起的局部組織壞死，而詳細的機制仍需要進一步的探討。

The victims of cobra snake bite usually exhibit clinical symptoms with a systematic neurological abnormality such as muscle weakness/respiratory arrest and/or local tissue damages such as toe gangrene/severe necrotizing fasciitis. Interestingly, African cobra of N. nivea produces mainly neurological effect without significant tissue damage and Asian cobra of N. atra and N. kaouthia produce mainly local tissue damage with few neurological symptoms. In order to understand possible mechanisms responsible for their distinct effect, we perform proteomic investigation of cobra venoms from N. atra, N. kaouthia and N. nivea and correlate the proteomic profile with the cell toxicity assay.
Based on the reverse-phase HPLC, SDS-PAGE and mass spectrometry analysis, all three cobra venoms consist of high molecular weight enzymes, PLA2s (phospholipase A2) excepting N. nivea, and three fingers toxin of NTXs (neurotoxins) and CTXs (cardiotoxins or cytotoxins), but with different degree of abundance. Interestingly, we cannot find out any evidence about the presence of PLA2 in N. nivea venom by mass spectrometry identification compared to the other two Asian venoms exhibiting mainly necrotic effect. We therefore hypothesize that the PLA2 might play a role in cell toxicity contributing further enhancing the necrotic cell death in the bitten tissue area.
Venom-induced cell toxicity of HEK-293 is then monitored by MTT methods to shed light on the effect of venom compositional change. The IC50 for the venom from N. atra, N. kaouthia and N. nivea are found to be ~ 12.2, 13.5 and 17.4 g/ml, respectively. Interestingly, all N. atra, N. kaouthia and N. nivea venom induced both apoptotic and necrotic cell death, of which apoptosis is the major cell death pathway. Remarkably, our present study revealed that PLA2 is one of the key component responsible for elevated cellular toxicity induced by the crude venom of N. atra, N. kaouthia as compared to that of N. nivea. Accordingly, the involvement of PLA2 in the clinical symptom of local tissue necrosis caused by cobra snake bite are proposed as a potential future avenue of research.

ABBREVIATIONS 1
Chapter 1 INTRODUCTION 3
1.1. Overview of cobra-snake venoms: Definition, compositions, and characteristics 3
1.1.1. Definition 3
1.1.2. Compositions 3
1.1.3. Characteristics 4
1.2. Taiwanese Naja atra (NA) 8
1.3. Vietnamese Naja kaouthia 9
1.4. Southern Africa Naja nivea 10
1.5. Pathways of cell death: Apoptosis, and necrosis 11
1.5.1. Apoptosis 11
1.5.2. Necrosis 13
Chapter 2 MATERIALS AND METHODS 15
2.1. Chemical reagents 15
2.2. Venom samples 15
2.3. C18 reverse-phase HPLC fractionation 15
2.4. Venom quantification and SDS-PAGE analysis 16
2.5. Protein digestion and identification 16
2.6. PLA2 activity 17
2.7. Cell culture 17
2.8. Cell viability assay 17
2.9. Determination of half maximal inhibitory concentration (IC50) 18
2.10. Morphological studies 18
2.11. Cell death assay (Annexin V-FITC/PI double staining for apoptosis and necrosis detection by flow cytometry assay) 18
2.12. Statistical analysis 18
Chapter 3 RESULTS 20
3.1. Venomic characterization of three cobra venoms from Naja atra (Taiwan), Naja kaouthia (Vietnam) and Naja nivea (Southern Africa) species 20
3.2. Comparative venomics of three cobra venoms from Naja atra (Taiwan), Naja kaouthia (Vietnam) and Naja nivea (Southern Africa) species 23
3.2.1. Comparison of 3FTX composition 23
3.2.2. Phospholipases A2 comparison 25
3.2.3. Comparison of other components 26
3.3. Comparison of enzymatic activity of PLA2 of three cobra crude venoms 26
3.4. Comparison of cell viability and morphological changes induced by three kinds of cobra crude venoms in human embryonic kidney 293 cells 27
3.5. Comparison of cell death pathways induced by three kinds of cobra crude venoms in human embryonic kidney 293 cells 28
3.6. Comparison of the functional roles of PLA2, PDE and 5-NT in cell toxicity induced by three cobra crude venoms in human embryonic kidney 293 cells 29
Chapter 4 DISCUSSION 31
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