臺灣博碩士論文加值系統

癲癇症是一種神經傳導物質分泌不平衡所產生的神經性病變，症狀從幾乎無法偵測的病發、產生嚴重的肢體抖動至造成生命危險。目前癲癇症狀多以抗癲癇藥物進行控制以減緩發作的程度或是減少發生機率，但現今有三分之一的癲癇病患無法以抗癲癇藥物獲得有效的控制，過去研究也發現過量服用抗癲癇藥物會造成肝毒性，因此開發新型且安全的抗癲癇藥物是重要且急迫的。癲癇症狀主要來自神經與肌肉組織之間的反應異常，因此現今抗癲癇藥物的開發主要是利用活體的動物模式進行。斑馬魚具有高生產力、發育快速及具有與哺乳類動物類似的神經反應系統等特性的脊椎動物。過去研究指出癲癇會造成幼魚泳動的上升，因此本實驗主要利用監測儀器Danio Vision大量且快速的進行行為學分析並以此判斷癲癇的嚴重程度。在本實驗中我們利用戊四唑 (pentylenetetrazole) 在六天大的幼魚中建立癲癇的動物疾病模式並利用此平臺篩選抗癲癇藥物。實驗結果發現抗癲癇藥物及維他命皆可減緩癲癇造成幼魚泳動上升的情形。實驗結果也指出苯妥因(Phenytoin)和左乙拉西坦(Levetiracetam)與維他命的同時處理及乙苯嘧啶二酮(Primidone)在維他命的預先處理時比起藥物單獨處理更可降低癲癇所誘發泳動上升的情形。另外我們也利用此平臺對66種中草藥萃取物進行篩選，我們找到編號317藥物-白芍可降低癲癇引發的泳動上生的情形，而在維他命與317藥物的預先處理後有更顯著的效果，但是以317藥物的主成分預先處理中未看到顯著的效果。未來我們希望藉由戊四唑誘發癲癇並以Danio Vision監測的模式大量且快速的篩選未知藥物的安全性以及對癲癇症狀治療的能力。

Epilepsy is a neurological disorder about imbalanced neurotransmitters. The symptoms of epilepsy could be diverse from brief and nearly undetectable, vigorous body shaking to life-threatening. Patients with epilepsy are usually prescribed with anti-epileptic drugs (AEDs) to ease or prevent epilepsy. However, there are still one third of the patients with epilepsy cannot be relieved through AEDs treatment. Previous study showed that overdose with AEDs may result in liver toxicity. Therefore, developing a new and safe AED is an important and urgent issue. The action of epilepsy seizure is induced by neuromuscular disorders. AEDs are mainly screening in animal models. Zebrafish is a vertebrate with high productivity, quickly development and comparable neurological system with mammals. Previous studies indicated that increased moving distance is observed in larvae with epileptic seizure. A high-throughput tracking system, Danio Vision, was used in this study to perform the behavioral analysis and evaluate the severity of the seizure. Pentylenetetrazole (PTZ) was treated in 6 day-post-fertilization (dpf) larvae to establish the model of epileptic seizure and screen AEDs. The results showed that both AEDs and vitamin relieve the increased moving distance in PTZ-treated larvae. The result also showed that Phenytoin and Levetiracetam co-treated with vitamin mixture and primidone with vitamin pre-treatment decrease the induced moving distance in PTZ-treated larvae. Furthermore, 66 herbal extracts were screened in this platform, and found that No. 317, Paeonia lactiflora decrease the increased moving distance in PTZ-treated larvae. In addition, larvae pre-treated with vitamin mixture and No. 317 exhibit a better rescued effect than vitamin mixture or No. 317 alone. However, no significantly rescued effect was observed in larvae pre-treated with the main ingredient of 317. In the future, we can use the disease model of PTZ-induced seizure and analyzed by Danio Vision as the high-throughput drug screening platform to estimate new therapeutic strategies or identify the new and safe AEDs

摘要……………………………………………………………………………………………………i
Abstract………………………………………………………………………………………ii
Acknowledgement…………………………………………………………………iii
Table of Content…………………………………………………………………iv
List of Figure………………………………………………………………………vi
List of Table………………………………………………………………………vii
Abbreviations……………………………………………………………………viii
I. Introduction……………………………………………………………………………1
1. Epilepsy………………………………………………………………………………………1
1.1. The causes and impacts of epilepsy…………………1
1.2. The prevalence of epilepsy………………………………………1
1.3. Current treatment of epileptic seizure………1
2. Functions of clinical used AEDs…………………………2
Valproic acid (VPA)………………………………………………………………………………2
Phenytoin (PHT)…………………………………………………………………………………………3
Primidone (PMD)…………………………………………………………………………………………3
Lamotrigine (LMT)……………………………………………………………………………………3
Levetiracetam (LEV)………………………………………………………………………………4
3. Current models of epileptic seizure………………4
4. Zebrafish (Denio rerio)………………………………………………5
5. Vitamin supplement……………………………………………………………5
Vitamin D…………………………………………………………………………………………………………6
Vitamin E…………………………………………………………………………………………………………7
Co-enzyme Q10………………………………………………………………………………………………7
Vitamin B6………………………………………………………………………………………………………8
Vitamin B9………………………………………………………………………………………………………8
II. Rationale……………………………………………………………………………………9
III. Specific aims………………………………………………………………………10
IV. Material and methods……………………………………………………11
1. Fish line and maintenance………………………………………11
2. Drug treatment……………………………………………………………………11
3. Herbal extracts preparation…………………………………12
4. Behavior recording and analysis: Danio Vision……12
5. Statistic analysis…………………………………………………………13
V. Results and figures………………………………………………………14
1. PTZ- and GT-induced seizure models in zebrafish………………………………………………………………………………………………………14
2. AEDs relieved the PTZ-induced seizure in zebrafish………………………………………………………………………………………………………16
3. Vitamin mixture can decrease high moving distance caused by seizure…………………………………………………………………………………18
4. Vitamin mixture decrease the survival rate of PTZ and VPA co-treated larvae……………………………………………………………20
5. Vitamin B6, pyridoxine, decrease the survival rate of PTZ and VPA co-treated larvae……………………………22
6. Co-treated vitamin mixture enhanced the rescue effect of PHT and LEV in PTZ-treated larvae……………25
7. Pre-treat with vitamin mixture could reduce the seizure performance, and it could improve PMD treatment………………………………………………………………………………………………………30
8. Screen for herbal extracts which are safe and without seizure-like activity…………………………………………………34
9. The moving distance was decreased in No. 317 and PTZ co-treated larvae but it was no enhancement in Vit.s co-treatment………………………………………………………………………………………………34
10. No. 317 pre-treatment could decrease the moving distance in PTZ-treated larvae and it could be enhanced in addition of Vit.s…………………………………………………………………………36
11. Paeoniflorin (PNF) pre-treatment could not decrease the moving distance in PTZ-treated larvae………………………………………………………………………………………………………………38
VI. Discussions……………………………………………………………………………40
VII. Table……………………………………………………………………………………………47
VIII. References………………………………………………………………………………50
IX. Appendices………………………………………………………………………………55

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