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研究生：	宋伊婷 Sung, Yi-Ting
論文名稱：	血栓素及血栓素受體訊息在短期與長期被動吸菸造成動脈血栓性疾病及肺部損傷之角色 The Role of Thromboxane A2/Thromboxane Receptor Signaling in Acute and Prolonged Passive Smoking Induced Arterial Thrombotic Disease and Pulmonary Injuries
指導教授：	鄭劍廷 Chien, Chiang-Ting
學位類別：	碩士 Master
系所名稱：	生命科學系 Department of Life Science
論文出版年：	2016
畢業學年度：	104
語文別：	英文
論文頁數：	93
中文關鍵詞：	二手菸、三手菸、血管栓塞、肺部損傷、血栓素、血栓素受體
英文關鍵詞：	Second-hand smoke, Third-hand smoke, Thrombosis, Pulmonary Injury, Thromboxane A2, Thromboxane receptor
DOI URL：	https://doi.org/10.6345/NTNU202204788
論文種類：	學術論文
相關次數：	點閱：117 下載：14
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被動菸害，包含二手菸害及三手菸害，其中二手菸害又分成主流菸煙及側流菸煙，被動菸害與主動菸害皆會造成心血管疾病及肺部疾病，本研究主要利用側流菸煙及三手菸探討心血管及肺部發炎性相關疾病。
香菸中的活性氧化物質會導致血小板過度活化，促進血栓形成，而花生四烯酸經血栓素合成酶 (Thromboxane A2 synthase, TXAS) 合成血栓素A2 (Thromboxane A2, TXA2) ，與血栓素受體 (Thromboxane prostanoid receptor, TP receptor) 結合會引起血小板活化，而血小板過度活化或內皮細胞受損，又會導致TXA2濃度增加，然而過去對於香菸及TXAS-TXA2-TP 訊息傳遞路徑的調控仍未釐清。
本研究利用TXAS、TP基因剔除小鼠及兩者基因皆剔除之小鼠，吸入側流菸煙及三手菸煙與動脈血栓性疾病的模式，探討被動菸害對動脈血栓形成及肺部發炎性變化。實驗分成TXAS+/+TP+/+、TXAS-/-TP+/+、TXAS+/+TP-/- 及TXAS-/-TP-/-小鼠並給予無菸煙暴露，菸煙暴露1週，菸煙暴露5週及菸煙暴露8週處理，共16組（各組n=6），並利用氯化鐵誘發急性動脈血管栓塞以檢測血栓形成所需時間，再藉由病生理及分生結果了解香菸及TXAS-TXA2-TP引起肺部發炎性之關聯。我們利用菸煙暴露刺激以評估肺損傷與氯化鐵誘發之急性動脈血管栓塞在四種小鼠之作用。我們以化學發光放大方式偵測活性氧數量，以西方墨點和免疫染色法探究氧化壓力、細胞凋亡、細胞自噬、發炎性細胞凋亡和發炎之分生機轉。
研究結果指出利用氯化鐵誘發之急性動脈血管栓塞，其血栓形成時間之結果在不同基因老鼠與菸煙處理之間，並無顯著關聯性，表示各基因對於菸煙暴露時間的反應具有一致性，但菸害仍會影響血栓形成，因而利用血小板吸附模式探討不同基因鼠的結果，在無菸害暴露的老鼠裡發現TXAS+/+TP+/+鼠之血小板吸附性顯著顯著高於其他三個品系品的老鼠，未來會再加入抽煙處理的組別以繼續探討;在肺部發炎性疾病方面，由病生理結果發現，不同基因鼠與菸煙處理之間具顯著關聯，表示菸煙會藉由TXAS-TXA2-TP訊息傳遞路徑引起肺部發炎性疾病。

Passive smoking contains second-hand smoke (SHS) and third-hand smoke (THS). SHS can be divided into mainstream smoke and sidestream smoke. Both active and passive smoking would cause cardiovascular disease (CVD) and pulmonary injuries. We used sidestream smoke and THS as our cigarette smoke (CS) model.
Oxidants in cigarettes activate platelets consequently induce thrombus formation. It is well known that thromboxane A2 (TXA2) through its receptor—thromboxane prostanoid (TP) receptor — would activate platelets, and the overactivation of platelets or endothelial cells injury would further induce TXA2 generation. The relationship between CS and thromboxane A2 synthase (TXAS)-TXA2-TP signaling is still ambiguous.
In order to investigate the influence of CS on TXAS-TXA2-TP signaling in thrombosis and pulmonary injuries, we used TXAS and TP gene knockout mice, inhaled sidestream and THS as whole body CS exposure model. Mice were divided into TXAS+/+TP+/+, TXAS-/-TP+/+, TXAS+/+TP-/- and TXAS-/-TP-/- treated with non-CS exposure (i.e. Control), acute cigarette smoke exposure group (ACE), prolonged cigarette smoke exposure 5 weeks group (PCE 5) and prolonged cigarette smoke exposure 8 weeks group (PCE 8) (16 groups, n=6 in each group). We evaluated the effects of CS exposure on lung injuries and FeCl3-induced thrombus formation in these four groups. We utilized the chemiluminescence amplification method to measure the amount of reactive oxygen species (ROS), western blot and immunohistochemistry to explore the underlying mechanisms including oxidative stress, apoptosis, autophagy, pyroptosis and inflammation in these animals.
Our results showed CS exposure triggered the formation of thrombus by the index of time to occlusion (TTO). The response of CS shortened TTO was attenuated by blocking TXAS or TP receptors. CS exposure induced oxidative stress would cause pulmonary injuries including endothelial dysfunction, endoplasmic reticulum (ER) stress, inflammation, cell apoptosis, autophagy and pyroptosis.
In conclusion, TXAS-TXA2-TP signaling plays an important role in CS induced pulmonary injury and FeCl3-induced thrombus formation.

I.    中文摘要    4
II.    ABSTRACT    6
III.    ABBREVIATIONS    8
IV.    INTRODUCTION    12
1. THE ENVIRONMENTAL TOBACCO SMOKE AND THIRDHAND SMOKE    12
1.1 MAINSTREAM AND SIDESTREAM SMOKE OF ENVIRONMENTAL TOBACCO SMOKE    12
1.2 THIRDHAND SMOKE    12
2. CIGARETTE SMOKE (CS) EXPOSURE INCREASES THE RISK OF THROMBOTIC CARDIOVASCULAR DISEASE    13
3. CIGARETTE SMOKE EXPOSURE LEADS TO PULMONARY INJURY    14
4. THE ASSOCIATION OF TXA2-TXAS-TP SIGNALING AND THE PATHOPHYSIOLOGY OF THORMBOSIS AND PULMONARY INJURY    15
4.1. SMOKING INCREASES PLATELET ACTIVATION AND THROMBOSIS PARTIALLY THROUGH ACTIVATING TXAS-TXA2-TP SIGNALING PATHWAY    16
4.2 SMOKING LEADS TO PULMONARY INJURY PARTIALLY THROUGH TXAS-TXA2-TP SIGNALING PATHWAY    17
5. USING TXAS OR TP GENE KNOCKOUT MICE FOR EXPLORING TXAS-TXA2-TP SIGNALING PATHWAY    17
6. AIM AND PURPOSE    18
V.    MATERIALS AND METHODS    19
1. ANIMALS    19
1.1 TXAS GENE KNOCKOUT MICE    19
1.2 TP GENE KNOCKOUT MICE    19
1.3 TXAS AND TP DOUBLE KNOCKOUT MICE:    20
1.4 GROUPING    20
1.5 SURGICAL PREPARATION    21
2. FECL3-INDUCED ACUTE ARTERIAL THROMBOSIS    21
3. CIGARETTE SMOKE EXPOSURE PROTOCOL    22
4. WHOLE BLOOD REACTIVE OXYGEN SPECIES DETECTION    22
5. BRONCHOALVEOLAR LAVAGE FLUID REACTIVE OXYGEN SPECIES DETECTION    22
6. IMMUNOHISTOCHEMISTRY (IHC)    23
7. TERMINAL DEOXYNUCLEOTIDE TRANSFERASE DUTP NICK END LABELING STAIN    24
8. WESTERN BLOT    26
9. PLATELET ADHESIVENESS DETECTION    27
10. MALONDIALDEHYDE DETECTION ASSAY    28
VI.    RESULTS    30
1. ACUTE ARTERIAL THROMBOSIS MODEL OF TIME TO OCCLUSION    30
2. PLATELET ADHESIVENESS DETECTION IN MESENTERIC ARTERIES    30
3. WHOLE BLOOD REATVIE OXYGEN SPECIES DETECTION    31
4. BRONCHOALVEOLAR LAVAGE FLUID OF REACTIVE OXYGEN SPECIES    31
5. MALONDIALDEHYDE CONCENTRATION DETECTION    32
6. HEMATOXYLIN AND EOSIN (H&E) STAIN    33
7. IMMUNOHISTOCHEMISTRY ANALYSIS    34
8. TERMINAL DEOXYNUCLEOTIDE TRANSFERASE DUTP NICK END LABELING STAIN    37
9. WESTERN BLOT    37
VII.    DISCUSSION    43
1.    CIGARETTE SMOKE EXPOSURE PROMOTE THROMBOSIS AND PULMONARY INJURIES    43
2. THE RELATIONSHIP BETWEEN CIGARETTE SMOKE EXPOSURE AND TXAS-TXA2-TP SIGNALING PATHWAY IN THROMBOSIS    43
3.    THE RELATIONSHIP BETWEEN CIGARETTE SMOKE EXPOSURE AND TXAS-TXA2-TP SIGNALING IN PULMONARY INJURIES    44
VIII.    CONCLUSION    49
IX.    REFERENCES    50
X.    FIGURES AND TABLES    55
TABLE.1 TIME TO OCCLUSION OF CAROTID ARTERY IN FOUR GENOTYPE MICE WITH FOUR DIFFERENT PERIODS OF CIGARETTE SMOKE EXPOSURE    55
FIGURE. 1 WHOLE-BODY SMOKE EXPOSURE MODEL    56
FIGURE. 2 THE CAROTID ARTERY BLOOD FLOW OF CONTROL GROUP IN THE FOUR GENOTYPES MICE    57
FIGURE. 3 THE CAROTID ARTERY BLOOD FLOW OF ACE GROUP IN THE FOUR GENOTYPES MICE    58
FIGURE. 4 THE CAROTID ARTERY BLOOD FLOW OF PCE 5 TREATED GROUP IN THE FOUR GENOTYPES MICE    59
FIGURE. 5 THE CAROTID ARTERY BLOOD FLOW OF PCE 8 TREATED GROUP IN THE FOUR GENOTYPES MICE    60
FIGURE. 6 COMPARISONS OF TIME TO OCCLUSION OF CAROTID ARTERY IN MICE BASED ON CIGARRETTE SMOKE EXPOSURE PERIODS AND GENOTYPES    61
FIGURE. 7 PLATELET ADHESIVENESS IN MESENTARIC ARTERY OF DIFFERENT GENOTYPE MICE    64
FIGURE. 8 COMPARISONS OF LUMINOL-INDUCED BLOOD H2O2 ACTIVITES IN MICE BASED ON CIGARETTE SMOKE EXPOSURE PERIODS AND GENOTYPES    65
FIGURE. 9 REACTIVE OXYGEN SPECIES LEVELS IN BRONCHOALVEOLAR LAVAGE FLUID    66
FIGURE. 10 PLASMA MDA CONCENTRATION    67
FIGURE. 11 PULMONARY ARTERY THICKNESS HISTOLOGICAL FETURE AND RELATIVE THICKNESS    68
FIGURE. 12 HISTOLOGICAL FETURE OF LUNG    71
FIGURE. 13 IMMUNOHISTOCHEMISTRY OF 4-HNE    72
FIGURE. 14 IMMUNOHISTOCHEMISTRY OF VWF    74
FIGURE. 15 IMMUNOHISTOCHEMISTRY OF IL-1Β    76
FIGURE. 16 IMMUNOHISTOCHEMISTRY OF PARP-1    78
FIGURE. 17 IMMUNOHISTOCHEMISTRY OF BECLIN-1    80
FIGURE. 18 TERMINAL DEOXYNUCLEOTIDE TRANSFERASE DUTP NICK END LABELING STAIN    82
FIGURE. 19 WESTERN BLOT OF PARP-1    84
FIGURE. 20 WESTERN BLOT OF BCL-2 AND BAX    86
FIGURE. 21 WESTERN BLOT OF LC3 Β    87
FIGURE. 22 WESTERN BLOT OF BECLIN-1    88
FIGURE. 23 WESTERN BLOT OF 4-HNE    89
FIGURE. 24 WESTERN BLOT OF VWF    90
FIGURE. 25 WESTERN BLOT OF NF-ΚB    91
FIGURE. 26 WESTERN BLOT OF ATF-6    92
FIGURE. 27 WESTERN BLOT OF P-JNK    93
                                

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