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系統識別號 U0026-1901201514431400
論文名稱(中文) 急性血管栓塞中Rho激酶蛋白和相關訊息傳遞路徑所扮演的角色
論文名稱(英文) Roles of ROCK (Rho-associated protein kinase) and its Associated Pathways During Acute Vascular Thrombosis Events
校院名稱 成功大學
系所名稱(中) 臨床醫學研究所
系所名稱(英) Institute of Clinical Medicine
學年度 103
學期 1
出版年 104
研究生(中文) 王裕文
研究生(英文) Yu-Wen Wang
學號 S96011061
學位類別 碩士
語文別 英文
論文頁數 51頁
口試委員 指導教授-劉秉彥
口試委員-陳品晟
口試委員-林寶彥
中文關鍵字 急性心肌梗塞  凝血酶  巨噬細胞  Rho激酶  半乳糖凝集素-3 
英文關鍵字 acute myocardial infarction  thrombin  macrophages  galectin-3  ROCK 
學科別分類
中文摘要 急性心肌血管栓塞是主要造成世界人類死亡的心血管疾病之一。而急性心肌梗塞發作是由凝血酶刺激生成的血栓堵塞至冠狀動脈所造成。在心血管栓塞疾病早期中,巨噬細胞已被證實部份藉由Rho激酶蛋白以及其相關訊息傳遞路徑刺激心血管動脈粥狀硬化以及促進血管斑塊生成。然而,在冠狀動脈血管壁破裂形成急性血管栓塞過程中,Rho激酶蛋白是如何調控巨噬細胞功能,以及其相關訊息傳遞路徑,目前還不是很清楚。先前研究指出,半乳糖凝集素(Galectin)家族中,半乳糖凝集素-3 (galectin-3)會促進巨噬細胞遷移和其吞噬作用。因此galectin-3可能在凝血酶刺激巨噬細胞和Rho激酶活化機制中扮演重要角色。在我們的研究中,我們希望能了解在活化巨噬細胞中Rho激酶和galectin-3機制上的關連性;並且了解藉由這條訊息傳遞路徑,來提供未來新的治療血管栓塞的標的。
為了探究活化巨噬細胞Rho激酶表現、活性以及galectin-3的分泌,我們利用凝血酶來刺激源於THP-1之巨噬細胞株。首先,我們藉由西方點墨法來探討凝血酶刺激巨噬細胞後, Rho激酶相關路徑蛋白和galectin-3分泌的關連性。接著,我們使用Rho激酶的抑制劑來研究活化之巨噬細胞galectin-3的分泌是否會有影響。然後,我們使用凝血酶來刺激巨噬細胞,並利用流式細胞儀偵測巨噬細胞上的M1 marker 和M2 marker。藉此,了解凝血酶刺激後會偏向M1還是M2的型態。除此之外,我們同時偵測凝血酶刺激後的巨噬細胞的移動能力是否會有所改變。最後,我們也比較急性心肌梗塞病人的巨噬細胞中galectin-3和Rho激酶的下游產物pERM的表現是否和正常人有差異。
我們發現在凝血酶刺激巨噬細胞24小時後,galectin-3分泌活化量最大。接著,在凝血酶刺激固定時間後,我們也發現Rho訊息傳遞相關蛋白表現也有提高的表現。在Rho激酶抑制劑處裡下,凝血酶刺激後的巨噬細胞之galectin-3分泌量會受到阻遏。在細胞功能檢測方面,我們發現在凝血酶刺激後,巨噬細胞更會傾向M1型態。此外凝血酶刺激後的巨噬細胞的移動能力也比較強。最後在檢體實驗中,我們發現急性心肌梗塞病人的巨噬細胞中galectin-3以及Rho激酶的下游產物pERM表現比正常人高。
在此我們發現,經凝血酶刺激可促使巨噬細胞分化成M1發炎形態並強化巨噬細胞移動能力,且也能促進巨噬細胞Rho激酶的表現進而使galectin-3分泌。
英文摘要 Acute myocardial infarction (AMI) is a leading cause of death of cardiovascular disease (CVD) in the world. It can be triggered by thrombin, which promotes thrombosis and causes coronary artery occlusion. In early CVD, monocyte and macrophage play major role in atherosclerosis and promote plaque formation partially via Rho kinase (ROCK) signal pathway. However, it remained unknown how ROCK signaling or its associated pathways play the role on monocytes and macrophages functions during the AMI development process. Previous studies have shown that galectin-3 could promote inflammation cell migration and macrophage phagocytosis. It is possible that galectin-3 may play as the mechanism between activation of macrophages or monocytes under thrombin stimulation thus facilitate ROCK activation. Therefore, our aim is to test the interaction between galectin-3 and Rho protein signal pathway in macrophages.
To investigate the ROCK activity, ROCK expression and galectin-3 secretion in macrophages, thrombin was used for the activation of THP-1 macrophage cell line. After stimulation of thrombin in macrophages, we studied the relationship between ROCK and galectin-3 secretion by western blots. Then, we tested the galectin-3 secretion among macrophage with ROCK inhibitor after thrombin stimulation. Flow cytometry and migration assay were applied to detect the M1/M2 macrophage expression distribution and migration functional changes after thrombin treatment. Finally, to prove that ROCK is important in human acute thrombotic event, we detected the phosphor-ezrin/radixin/moesin (pERM), which are the downstream molecules of ROCK, and galectin-3 expression in macrophages isolated from AMI patient and also healthy donors.
We found that galectin-3 secretion was activated in macrophage after thrombin treatment peaking at 24 hours. Interestingly, Rho-associated pathway protein expressions increased after thrombin treatment earlier at 12 hours. After Rho-kinase inhibitor treatment, the expression of galectin-3 decreased in the activated macrophage. Regarding functional assay, more percentage of THP-1-derived macrophages were prone to become M1 type macrophage after thrombin treatment in flow cytometry and had greater migration ability after thrombin treatment. Finally, we proved that pERM and galectin-3 expression in AMI patient’s macrophages were higher than healthy donor’s macrophages.
In conclusion, thrombin treatment can stimulate macrophage to M1 type and their migration ability, thus promote Rho-kinase signal pathway expression and galectin-3 secretion.
論文目次 Chinese abstract 1
English abstract 2
Abbreviations 6
Chapter 1 Introduction 8
1.1 Acute myocardial infarction (AMI) is triggered by thrombin-induced inflammation 8
1.2 Macrophages and thrombin 9
1.3 Rho-associated coiled-coil containing protein kinases (ROCKs) and thrombosis 9
1.4 Galectin-3 and cell mobilization function 10
1.5 Hypothesis 11
1.6 Specific aims 12
Chapter 2 Material and Methods 13
2.1 Material 13
2.2 Cell culture and isolation of artery blood mononuclear cells 13
2.3 Cell lysate protein and secreted protein collection 14
2.4 Bicinchoninic acid (BCA) protein assay 15
2.5 Conditions of thrombin stimulation and immunoblots 15
2.5.1 Time-dependent stimulation 15
2.5.2 Dose-dependent stimulation 16
2.5.3 ROCK signaling pathway protein expression 17
2.5.4 The protein expression after inhibition of ROCK 18
2.5.5 The protein expression of human primary mononuclear cells 19
2.6 Flow cytometry 20
2.7 Migration assay 21
Chapter 3 Results 23
3.1 Thrombin stimulation of galectin-3 in macrophages 23
3.2 ROCK activation is important for thrombin stimulation of galecin-3 in macrophages 24
3.3 Macrophages are prone to M1 type after thrombin treatment 25
3.4 Macrophages enhance their migration ability after thrombin treatment 26
3.5 ROCK activity and galectin-3 had higher expression in AMI patient macrophages 26
Chapter 4 Discussion 28
Conclusion 30
Tables 31
Figures 33
References 49
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