臺灣博碩士論文加值系統

心血管疾病包括冠心病、心絞痛、急性心肌梗塞等等，為世界死因之首。根據台灣衛生福利部的統計，我國心血管疾病為第二大死因，每十萬人就有88.1人於心血管疾病，僅次於惡性腫瘤的199.6人。心肌缺血會使心肌細胞發生強烈的發炎反應且再灌流後會使冠狀動脈釋放出大量活性氧物質，而活性氧物質又促進血栓的形成，影響到血栓素合成酶(Thromboxane A2 synthase, TXAS)-血栓素 (Thromboxane A2, TXA2)-血栓素受體 (Thromboxane prostanoid receptor, TP receptor)訊息傳遞路徑，包括增加了血栓素合成酶(Thromboxane A2 synthase, TXAS)的表達和血栓素受體(Thromboxane prostanoid receptor, TP receptor)的活性，最終增加了血管內皮素(Endothelin-1, ET-1)釋放造成更加嚴重的傷害。
本篇研究即是要探討抑制掉TXAS-TXA2-TP訊息傳遞路徑後，ET-1所喚起的傷害和在心肌缺血再灌流的傷害上扮演著什麼樣的角色。我們使用三種不同基因型的老鼠，TXAS+/+TP+/+、TXAS-/-TP+/+及TXAS-/-TP-/-小鼠並將實驗分為以下幾組。
1. 所有的小鼠將被隨機靜脈注射生理實驗水、U46619 (TXA2 agonist, 2 mg/kg)和ET-1(0.001-0.2 µg/kg)測量其心臟微循環，共四組(各組N=6)。
2. 所有的小鼠將被隨機執行血管環模型實驗(wire myography)，測量其血管對藥物(norepinephrine, acetylcholine, U46619, ET-1 )的收縮或舒張反應，共四組(各組N=6)。
3. 所有的小鼠將被隨機執行心肌缺血再灌流模型(myocardial ischemia/reperfusion model)手術，共四組(各組N=6)。
並搭配組織免疫染色、血漿心肌旋轉蛋白(Troponin I)含量測定來看探討其細胞凋亡、細胞自噬、發炎性細胞凋亡和發炎之分生機轉及心肌梗塞之嚴重程度。
我們的研究結果指出抑制TXAS-TXA2-TP訊息傳遞路徑，可以有效的減少心肌缺血再灌流所引起的細胞凋亡、發炎性細胞凋亡和氧化壓力，對小鼠具有較佳之心臟保護效果。

Cardiovascular disease has become one of the most harmful human diseases with highest morbidity in the world, especially coronary heart disease and myocardial infarction. According to Ministry of Health and Welfare, the cardiovascular disease is the second cause of death and accounts for 0.881‰ death per year in Taiwan, second only to cancer.
Myocardial ischemia/reperfusion (I/R) induces the release of oxidants in coronary arteries. Following the production, the oxidants may activate platelets and consequently induce thrombus formation. It is well known that thromboxane A2 synthase (TXAS) —thromboxane A2 (TXA2)—thromboxane prostanoid receptor (TP)— would activate TP, and increasing release of endothelin-1 (ET-1) to bring about more serious injury.
In order to explore the role of TXAS-TXA2-TP pathway in endothelin-1 (ET-1) activation during I/R injury, we utilized mouse with gene depletion in TXAS (TXAS–/–) and both TXAS and TP (TXAS–/–TP–/–) mice. All mice were randomly subjected to intravenous normal saline, endothelin-1 (0.001-0.2 µg/kg body weight), U46619 (TXA2 agonist, 2 mg/kg body weight). Using Wire myograph model to determine the vascular reactivity of rat mesentery arteries, we investigated the possible signaling pathway between ET-1 and TXAS-TXA2-TP. In myocardial I/R model, the cardiac injuries were evaluated by microcirculation, electrocardiogram and plasma troponin I. We explored the mechanisms including apoptosis, pyroptosis and inflammation via level of plasma troponin I and immunohistochemistry stain in these animals.
Our results indicate that the inhibition of TXAS-TXA2-TP pathway provides cardiac protection against myocardial I/R injury.

I. 中文摘要 4
II. ABSTRACT 6
III. ABBREVIATIONS 8
IV. INTRODUCTION 10
1. THE CARDIOVASCULAR DISEASES IN WORLDWIDE 10
2. MYOCARDIAL ISCHEMIA REPERFUSION INJURY 10
3. THE ASSOCIATION BETWEEN ENDOTHELIN-1 AND TXAS-TXA2-TP SIGNALING PATHWAY IN MYOCARDIAL ISCHEMIA REPERFUSION INJURY 11
4. USING TXAS OR TP GENE KNOCKOUT MICE FOR EXPLORING TXAS-TXA2-TP SIGNALING PATHWAY 14
5. AIM 14
V. MATERIALS AND METHODS 15
1. ANIMALS 15
1.1 WILD TYPE, TXAS AND TP GENE KNOCKOUT MICE 15
1.2 TXAS AND TP DOUBLE KNOCKOUT MICE: 16
1.3 SURGICAL PREPARATION 16
2. MEASUREMENT OF ELECTROCARDIOGRAPHIC PARAMETERS 17
3. CARDIAC MICROCIRCULATION DETERMINATION 17
4. WIRE MYOGRAPHY 18
5. INDUCTION OF ACUTE MYOCARDIAL INFRACTION 19
6. IMMUNOHISTOCHEMISTRY (IHC) 20
7. TERMINAL DEOXYNUCLEOTIDE TRANSFERASE DUTP NICK END LABELING STAIN 21
8. MEASUREMENT OF CARDIAC TROPONIN I 23
9. STATISTICAL ANALYSES 23
VI. RESULTS 24
1. HEART MICROCIRCULATION UNDER INTRAVENOUS NORMAL SALINE, U46619 AND ENDOTHELIN-1. 24
2. ECG UNDER INTRAVENOUS NORMAL SALINE, U46619 AND ENDOTHELIN-1. 25
3. EFFECT OF CONTRACTION AND RELAXATION IN MESENTERIC ARTERIES 26
4. MICROCIRCULATION AND ECG UNDER MYOCARDIAL ISCHEMIA REPERFUSION INJURY IN THREE GENOTYPE MICE 27
5. HEMATOXYLIN AND EOSIN (H&E) STAIN 28
6. TERMINAL DEOXYNUCLEOTIDE TRANSFERASE DUTP NICK END LABELING STAIN 28
7. IMMUNOHISTOCHEMISTRY ANALYSIS 29
8. PLASMA LEVEL OF TROPONIN I MEASUREMENT. 30
VII. DISCUSSION 31
1. PREVIOUS STUDIES OF TXAS-TXA2-TP SIGNALING AND ET-1 IN CARDIOVASCULAR DISEASES. 31
2. IMPACT OF ECG AND HEART MICROCIRCULATION THROUGH TXAS-TXA2-TP SIGNALING AND ET-1. 32
3. PHARMACOLOGICAL RESPOND IN RESISTANCE ARTERY THROUGH TXAS-TXA2-TP SIGNALING AND ET-1. 33
4. INHIBITION OF TXAS-TXA2-TP SIGNALING ATTENUATE INJURY EVOKED BY MYOCARDIAL ISCHEMIA REPERFUSION THROUGH APOPTOSIS, OXIDATIVE STRESS, INFLAMMATION AND PYROPTOSIS. 34
VIII. CONCLUSION 39
IX. REFERENCE 40
X. FIGURES AND TABLES 48
FIGURE. 1 ISCHEMIA/REPERFUSION MODEL IN MICE. 48
FIGURE. 2 CARDIAC MICROCIRCULATION UNDER INTRAVENOUS NORMAL SALINE IN A MINUTE. 49
FIGURE. 3 CARDIAC MICROCIRCULATION UNDER INTRAVENOUS U46619 (TP AGONIST, 2MG/KG) IN A MINUTE. 50
FIGURE. 4 CARDIAC MICROCIRCULATION UNDER INTRAVENOUS ET-1 (2.5 µG/KG) IN A MINUTE. 51
FIGURE. 5 CARDIAC MICROCIRCULATION UNDER INTRAVENOUS ET-1 (25 µG/KG) IN A MINUTE. 52
FIGURE. 6 CARDIAC MICROCIRCULATION UNDER INTRAVENOUS ET-1 (250 µG/KG) IN A MINUTE. 53
FIGURE. 7 (A) PERFUSION UNIT OF CARDIAC MICROCIRCULATION UNDER INTRAVENOUS NORMAL SALINE (RED ARROW) AMONG THREE GENOTYPE MICE. (B) THE PERCENTAGE OF PERFUSION UNIT AMONG MICE GENOTYPES. 54
FIGURE. 8 (A) PERFUSION UNIT OF CARDIAC MICROCIRCULATION UNDER INTRAVENOUS U46619 (TP AGONIST, 2MG/KG, RED ARROW) AMONG THREE GENOTYPE MICE. (B) THE PERCENTAGE OF PERFUSION UNIT AMONG MICE GENOTYPES. 55
FIGURE. 9 (A) PERFUSION UNIT OF CARDIAC MICROCIRCULATION UNDER INTRAVENOUS ET-1 (2.5µG/KG, 25 µG/KG AND 250µG/KG, RED, BLUE AND GREEN ARROW) AMONG THREE GENOTYPE MICE. 56
FIGURE. 10 ECG OF THREE GENOTYPE MICE UNDER INTRAVENOUS NORMAL SALINE IN THREE MINUTES. 57
FIGURE. 11 ECG OF THREE GENOTYPE MICE UNDER INTRAVENOUS U46619 (TP AGONIST, 2MG/KG) IN THREE MINUTES. 58
FIGURE. 12 FIGURE. 10 EKG OF THREE GENOTYPE MICE UNDER INTRAVENOUS ET-1(2.5, 25 AND 250 µG/KG). 59
FIGURE. 13 THE R-R INTERVAL UNDER INTRAVENOUS (A) NORMAL SALINE, (B) U46619 (2MG/KG) AND (C) ET-1 (2.5, 25 AND 250 µG/KG) AMONG THREE GENOTYPE MICE. 60
FIGURE. 14 EFFECT OF CONTRACTION AND RELAXATION IN MESENTERIC ARTERIES. (A) NOREPINEPHRINE (B) ACETYLCHOLINE (C) U46619 (D) ENDOTHELIN-1. 62
FIGURE. 15 CARDIAC MICROCIRCULATION (A) AND ECG (B) WITH MYOCARDIAL ISCHEMIA/REPERFUSION INJURY IN B6 MICE. 63
FIGURE. 16 CARDIAC MICROCIRCULATION (A) AND ECG (B) WITH MYOCARDIAL ISCHEMIA/REPERFUSION INJURY IN TXAS-/-TP+/+ MICE. 64
FIGURE. 17 CARDIAC MICROCIRCULATION (A) AND ECG (B) WITH MYOCARDIAL ISCHEMIA/REPERFUSION INJURY IN TXAS-/-TP-/- MICE. 65
FIGURE. 18 HISTOLOGICAL FEATURE OF HEART. 66
FIGURE. 19 TERMINAL DEOXYNUCLEOTIDE TRANSFERASE DUTP NICK END LABELING STAIN 67
FIGURE. 20 IMMUNOHISTOCHEMISTRY OF BECLIN-1 68
FIGURE. 21 IMMUNOHISTOCHEMISTRY OF IL-1Β 69
FIGURE. 22 IMMUNOHISTOCHEMISTRY OF 4-HNE 70
FIGURE. 23 PLASMA TROPONIN-I CONCENTRATION AFTER I/R. 71

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