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研究生:黃芝婷
研究生(外文):Chih-Ting Huang
論文名稱:重複腦部電擊對大白鼠大腦皮質基因表現調控的影響
論文名稱(外文):Identification of Differentially Expressed Messenger RNAs and Proteins in Rat Brain Induced by Repeated Electroconvulsive Shock
指導教授:陳嘉祥陳嘉祥引用關係
指導教授(外文):Chia-Hsiang Chen
學位類別:碩士
校院名稱:慈濟大學
系所名稱:人類遺傳研究所
學門:生命科學學門
學類:生物科技學類
論文種類:學術論文
論文出版年:2006
畢業學年度:94
語文別:英文
論文頁數:50
中文關鍵詞:大腦皮質基因電擊
外文關鍵詞:electroconvulsive shockBrain
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中文摘要
電氣痙攣治療是利用人為的方式在人類的腦部通過電流達到癲癇的效果,主要應用在醫治有重度憂鬱、躁症、精神分裂症或是其他精神疾病的患者。但目前對於電氣痙攣治療的治病機制卻是未知的。
本研究希望透過在對大白鼠使用重複的電療法後,利用二維電泳(2-Dimmesional Electrophoresis; 2-DE) 及Oligonucleotide Microarray分析、研究protein和messenger RNA在大白鼠大腦皮質表現的差異性以探討電療治療的作用機制。首先,用重複的Electroconvulsive Shock (ECS)法處理10隻Sprague Dawley公鼠 (45 mA,2 s,每天一次,連續14天),將兩個電極分別固定在公鼠的兩耳上;另外10隻對照組的Sprague Dawley公鼠處理的過程與實驗組的公鼠一樣,唯一的不同是對照組的公鼠沒有經過電流的處理。在2-DE的實驗,透過實驗結果比較對照組與實驗組老鼠的hippocampus蛋白質點之間的差異,並且將這些有差異的蛋白質點,利用質譜儀進行比對;比對結果發現有七個蛋白質,分別是quinoid dihydropteridine reductase, glutathione peroxidase (EC1.11.1.9) I protein, similar to BC023835 protein, similar to mitochondrial ribosomal protein L15, chaperonin containing TCP1 subunit 2 (beta), Ubiquilin 1, 與 similar to neuron navigator會受到電療治療的調控。其中,glutathione peroxidase (EC1.11.1.9) I protein則利用 Western Blot的方法加以驗證,發現這一個蛋白質表現在實驗組與對照組中並無明顯差異。
此外,從老鼠的frontal cortex抽出total RNA,利用microarray的方法分析基因表現的差異。在22,575個基因中,利用BRB-ArrayTools v3.3軟體分析,發現102個基因在實驗組與對照組中的mRNA表現是有顯著差異的。在這102個有顯著差異的基因中,挑選十個基因用 Real-Time Quantitative PCR的方法加以驗證。其中有4個基因證實在實驗組中的表現量與對照組是有顯著差異的 (p<0.05)。這四個基因分別為Dbi, Mgst3, S100B, 和S100A13。這些受電療治療調控的基因被認為與調控、傳導訊息及保護神經細胞相關。
Abstract
Electroconvulsive shock (ECS) therapy is an induction of artificial seizures by passing electricity through the patient’s brain. Electroconvulsive shock therapy is effective in treating people who have major depression, mania, schizophrenia, or other psychiatric disorders. The mechanism of action underlying the effectiveness of electroconvulsive shock remains unknown.
In this study, we aim to understand the mechanism of ECS by using two-dimensional gel electrophoresis (2-DE) and microarray analysis to identify differentially expressed genes in rat brains. We treated 10 male Sprague Dawley rats with repeated ECS, (45 mA, 2 s, once per day for 14 consecutive days), via ear-clip electrodes (UGO BASILE ECT UNIT 7801). Ten other male control rats were treated in the same way as the ECS-treated rats but with no electric current (sham treatment). In the 2-DE experiment, we identified several differentially expressed protein spots in the hippocampus of rats treated with ECS compared to control rats. These spots were analyzed by using mass spectrometry. seven identified proteins were quinoid dihydropteridine reductase, glutathione peroxidase (EC1.11.1.9) I pritein, similar to BC023835 protein, similar to mitochondrial ribosomal protein L15, chaperonin containing TCP1 subunit 2 (beta), Ubiquilin 1, and similar to neuron navigator. Expression of the glutathione peroxidase I was further confirmed by Western blot analysis. Unfortunately, the protein expression in ECS-treated and control rats was of no difference. As for the microarray experiment, we prepared total RNA from frontal cortex of rats; Microarray analysis was performed to detect differentially expressed genes. Furthermore, we used real-time quantitative PCR to confirm 10 out of 102 differentially expressed genes. Four genes showed significant difference between ECS-treated and control rats. They are Dbi, Mgst3, S100B, and S100A13.
This study explored some novel genes that were regulated by repeated ECS treatment. To conclude, those ECS-induced genes may have the functions related to neuromodulatory, neuroprotection and metabolic detoxication.
目錄(Content)
誌謝 II
中文摘要 III
Abstract V
1. Introduction 3
1.1. Electroconvulsive Shock Therapy 3
1.2. Relevant Publications 4
1.3. Microarray 5
1.4. 2-Dimensional Polyacrylamide Gel Electrophoresis (2-D PAGE) 6
2. Goal 8
3. Materials and Methods 9
3.1. Animals and ECS Therapy 9
3.2. Messenger RNA Expression 9
3.2.1. Total RNA Preparation 9
3.2.2. DNase Digestion 10
3.2.3. RNA Quality Assurance 11
3.2.4. Microarray Hybridization 11
3.2.5. Microarray Data Analysis 12
3.2.6. Reverse Transcription 13
3.2.7. Real-Time Quantitative PCR Conditions 13
3.2.8. Quantification and Normalization 14
3.2.9. Statistical Analysis 14
3.3. Proteomics 15
3.3.1. Protein Extraction and Quantification 15
3.3.2. Two-Dimensional Gel Electrophoresis 16
3.3.3. Silver Staining and Image Analysis 17
3.3.4. In-gel Digestion and Mass Spectrometric Analysis 18
3.3.5. Western Blot Analysis 18
4. Results 20
4.1. Microarray Results 20
4.1.1. Messenger RNA Expression Analysis with Agilent Microarray 20
4.1.2. Messenger RNA Expression Analysis with Real-Time Quantitative PCR 21
4.2. Proteomics Results 21
4.2.1. Identification of Differentially Expressed Proteins between Control and ECS by Mass Spectrometric Analysis 21
4.2.2. Validation of Differentially Expressed Proteins by Western Blot 22
5. Discussion 23
5.1. Microarray 23
5.1.1. Diazepam Binding Inhibitor (Dbi) 23
5.1.2. S100 Calcium-Binding Protein Beta (S100B) and Gene Similar to Human and Mouse S100A13 (S100A13) 24
5.1.3. Micorsomal Glutathione S-Transferase 3 (Mgst3) 25
5.2. Proteome 27
6. Summary 29
7. References 30
Appendices 35
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