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研究生：	林怡璇 Lin, Yi-Hsuan
論文名稱：	Galectin-3和Rho激酶在急性冠心症巨噬細胞上的交互作用研究 Interaction between Galectin-3 and Rho-associated protein kinase on macrophage during acute coronary syndrome
指導教授：	劉秉彥 Liu, Ping-Yen
學位類別：	碩士 Master
系所名稱：	醫學院 - 臨床醫學研究所 Institute of Clinical Medicine
論文出版年：	2021
畢業學年度：	109
語文別：	英文
論文頁數：	48
中文關鍵詞：	粥狀動脈硬化、急性冠心症、巨噬細胞、半乳糖凝集素-3 、Rho激酶、凝血酶
外文關鍵詞：	acute coronary syndrome, coronary thrombosis, galectin-3, ROCK
相關次數：	點閱：192 下載：0
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粥狀動脈硬化會導致血管斑塊的形成，經由不斷發炎、壞死、纖維化、血管鈣化的過程中，不穩定的血斑塊可能會破裂，此時免疫細胞浸潤與大量血小板聚集在斑塊破裂處形成血栓，血栓形成造成血流受阻，心肌供氧不足，最終可能會造成致命性的心肌壞死，我們稱之為急性冠心症後群(Acute coronary syndrome；ACS)。巨噬細胞會吞噬氧化型低密度脂蛋白(ox-LDL)，形成泡沫細胞(foam cell) 此為早期粥狀動脈硬化的病徵，大量的泡沫細胞累積在血管壁中，久而久累積處就會形成血斑塊，不穩定的血斑塊有大量免疫細胞浸潤，在血栓破裂的過程中，巨噬細胞扮演很重要的角色，巨噬細胞會移動到血管病變處造成血斑塊的不穩定，進而誘導血斑塊破裂，在我們的研究中，在病患進行經皮下導管治療時，我們採集病人冠狀動脈的血液進一步分析，發現急性冠心症的病患相較於慢性冠狀動脈疾病病患血清中會有較高的半乳糖凝集素-3 (galectin-3) ，進一步從血液中分離出來的外周血單個核細胞也有較高的galectin-3表現，除此之外，在急性冠心症病患的冠狀動脈血栓中也發現血栓上的巨噬細胞會有galectin-3的表現，為了在體外模擬巨噬細胞在急性冠心症情況下，THP-1所分化的巨噬細胞會加入凝血酶 (Thrombin) ，我們發現了加入thrombin後的巨噬細胞會分泌出更多的galectin-3並且移動的能力也會增強。在先前的研究報導顯示，thrombin可以刺激Rho激酶(ROCK)的活性，我們發現抑制了Rock的活性之後，巨噬細胞經由thrombin刺激所分泌的galectin-3會大量減少，因此，我們得知ROCK可以透過調節巨噬細胞galectin-3的分泌，進而影響巨噬細胞的移動能力，這可能會導致血斑塊的不穩定甚至是破裂。

Atherosclerosis leads to plaque formation at specific sites of the arteries through intimal inflammation, necrosis, fibrosis, and calcification. After decades of progress, plaques may suddenly cause life-threatening coronary thrombosis. Thrombus obstructs the blood flow and leads to lack of oxygen and nutrients for the heart caused acute coronary syndrome (ACS). Macrophages ingest ox-LDL to form foam cells and accumulate in the lesions is the initial symptom of atherosclerosis. In plaque ruptured process, macrophages will migrate to the lesions and secrete cytokine to make the plaque unstable. In our study, we collected patients’ blood from coronary arteries during percutaneous coronary intervention (PCI), and found that higher galectin-3 expression of serum and PBMC in ACS patients compared to chronic coronary syndrome (CCS) patients. Furthermore, galectin-3 expressed on macrophages in thrombus. Herein, we investigated how galectin-3 mediated in macrophages during ACS. THP-1 derived macrophages were treated with thrombin to mimic the microenvironment during ACS. After treated with thrombin, macrophages secreted more galectin-3 into medium, and increased the migration ability of itself. Thrombin is an inducer of Rho-associated kinase (ROCK). Inhibition of ROCK reduced the secretion of galectin-3 on macrophages. Through ROCK activated, increasing galectin-3 secretion of macrophages induced migration ability of macrophages. It may cause the plaque unstable even ruptured.

Abstract I
Chinese abstract II
誌謝III
Abbreviation VII
Chapter 1. Introduction 1
1.1	Coronary artery disease 1
1.2	Plaque formation 1
1.3	Acute coronary artery 2
1.4	Macrophages on acute coronary syndrome 2
1.5	Galectin-3 3
1.6	The expression of galectin-3 on atherosclerosis 3
1.7	Rho-associated protein kinase 4
1.8	Research motivation 4
1.9	Hypothesis 5
1.10 Specific Aims 5
Chapter 2. Materials and Methods 6
2.1 Clinical study design 6
Patients’ enrollment 6
Study Protocol 7
Immunofluorescent 7
2.2 Cell Culture 8
Human monocyte cell line 8
Reagents 8
Macrophage differentiation 9
Wound healing assay 9
Invasion assay 9
siRNA transfection 10
Enzyme-linked immunosorbent assay (ELISA) 11
RNA isolation and reverse transcription 11
Real-time quantitative PCR (RT-qPCR) 12
Protein extraction and Bicinchoninic acid (BCA) protein assay 12
Western blot assay 13
Zymography 13
2.3 Computer aid graphic design 14
Chapter 3. Results 15
3.1 Patients characteristic 15
3.2 Higher galectin-3 are expressed in ACS patients 16
3.3 THP-1 monocytes are differentiated into macrophages 16
3.4 Higher galectin-3 expression and secretion on macrophages after thrombin stimulated 17
3.5 Galectin-3 increases migration ability of macrophages 18
3.6 Galectin-3 can’t increase invasion ability of macrophages 18
3.7 Galectin-3 expression doesn’t affect the activity of ROCK 19
3.8 Inhibition of ROCK activity reduced the secretion of galectin-3 19
Chapter 4. Discussion 21
Chapter 5. Conclusion 23
References 24
Figures 28
Figure 1 The expression of galectin-3 was higher in the patients with ACS	29
Figure 2 THP-1 differentiated into macrophage by treated with PMA 31
Figure 3 Both secretion and cytosolic galectin-3 increased after treated with thrombin on THP-1 derived macrophages 32
Figure 4 Galectin-3 induced the migration ability of THP-1 derived macrophage 34
Figure 5 Galectin-3 won’t affect the invasion ability of THP-1 derived macrophage 36
Figure 6 ROCK activity was increased after thrombin stimulation on THP-1 derived macrophages 37
Figure 7 Inhibition of galectin-3 wouldn’t affect phosphorylation of MYPT-1 on THP-1 derived macrophages 38
Figure 8 Galectin-3 secretion was decrease on THP-1 derived macrophages after treated with ROCK inhibitor 40
Figure 9 Conclusion 41
Tables 42
Table 1. Primer sequences for PCR 42
Table 2. Characterization of patients with chronic coronary syndrome (stable) and acute coronary syndrome (acute) 43
Table 3. Lab parameters of patients with chronic coronary syndrome (stable) and acute coronary syndrome (acute) 44
Table 4. Medicines of patients of patients with chronic coronary syndrome (stable) and acute coronary syndrome (acute) 45
Supplementary figures 46
                                    

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2026-09-13公開

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