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研究生:簡含修
研究生(外文):JIAN, HAN-SIOU
論文名稱:探討疾病調節抗風濕藥物對泡沫細胞形成之影響
論文名稱(外文):To Investigate the Effect of Disease Modifying Anti-Rheumatic Drugs (DMARDs) on the Foam Cell Formation
指導教授:何令君
指導教授(外文):HO, LING-JUN
口試委員:陳安賴振宏何令君
口試委員(外文):CHEN, ANNLAI, JENN-HAUNGHO, LING-JUN
口試日期:2014-05-19
學位類別:碩士
校院名稱:國防醫學院
系所名稱:微生物及免疫學研究所
學門:生命科學學門
學類:微生物學類
論文種類:學術論文
論文出版年:2014
畢業學年度:102
語文別:中文
論文頁數:62
中文關鍵詞:動脈粥狀硬化類風溼性關節炎泡沫細胞
外文關鍵詞:AtherosclerosisRheumatoid arthritisfoam cellABCA1
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心血管疾病位居國人十大死因之一,而動脈粥狀硬化為心血管疾病形成的主因。動脈粥狀硬化是一種慢性且發炎性的心血管疾病,而在動脈粥狀硬化形成過程中,泡沫細胞扮演著相當重要的角色。巨噬細胞會藉由細胞表面上的清道夫接受器 (Scavenger Receptors, SRs)來吞噬氧化態低密度脂蛋白形成泡沫細胞,而細胞內多餘的膽固醇也會藉由膽固醇逆轉子(Reverse Cholesterol transporters, RCTs)排出細胞外,這兩個主要的機制調控著泡沫細胞的形成。過去的研究發現,在類風溼性關節炎的病人當中,有高達9.3%的患者有罹患心血管疾病,並認為堆積於類風溼性關節炎病人身體內的促發炎因子可能是造成動脈粥狀硬化早期形成的原因,而治療類風濕性關節炎的藥物能有效抑制發炎反應,因此,我們認為治療類風濕性關節炎的藥物或許可以有效降低泡沫細胞之形成。
在我們的研究當中選用了兩大類藥物,分別為疾病調節抗風濕藥物(Disease-Modifying Antirheumatic Drugs,DMARDs):Leflunomide、Azathioprine、Cyclosporine A、Hydroxychloroquine、Methotrexate、Sulfasalazine;以及類固醇藥物(steroidal anti-inflammatory drugs):Prednisolone、Dexamethasone來做研究。並觀察這八種藥物對THP-1巨噬細胞上SRs (SR-A和CD36)以及RCTs (ABCA1和ABCG1)表現、吞噬ox-LDL的能力以及總膽固醇含量來做其可能降低泡沫細胞形成之評估。
初步研究結果顯示,Leflunomide會隨著藥物的濃度增加而增加THP-1-macrophage上ABCA1的蛋白質表現量,這個結果推測Leflunomide可能會增加cholesterol efflux的功能。我們也發現,Cyclosporine A會降低SR-A的表現量、ox-LDL的吞噬以及總膽固醇的含量。此外,Hydroxychloroquine會增加ABCA1、ABCG1的表現量。因此我們認為Leflunomide、Cyclosporine A以及 Hydroxychloroquine可能具有降低泡沫細胞形成的潛力。
然而在使用刺激類固醇類藥物(Prednisolone和Dexamethasone)實驗中發現,這兩種藥物皆降低了THP-1-macrophage上ABCA1的表現量,顯示其可能增加泡沫細胞的生成,也提醒臨床上對於這類病人在使用類固醇類藥物時須特別小心。
Atherosclerosis is a chronic inflammatory disease of the wall of large- and medium-sized arteries and is a major health concern in the world. The formation of foam cells is a crucial reason in the process of atherosclerosis which start from the uptake of oxidized low-density lipoprotein (ox-LDL) by macrophage via scavenger receptors (SRs). In addition to accumulate lipid and activate endothelial cells, these foam cells release pro-inflammatory cytokines which further enhance the severity of the disease. Interestingly, regulation of foam cells depends on ingestion of ox-LDL as well as efflux of excess cholesterol via reverse cholesterol transporters (RCTs).
It has been showed that overall cardiovascular disease (CVD) had a lifetime prevalence (9.3%) among rheumatoid arthritis (RA) patients. The cumulative inflammation of RA, with the abundant synthesis of proinflammatory cytokines, may contributes directly to the early formation of the atheromatic plaque. Since disease modifying anti-rheumatic drugs (DMARDs) have strong anti-inflammatory effects, we planned to examine whether currently used DMARDs in RA patients are able to slow down the progression of foam cell formation.
We evaluated six DMARDs, including Leflunomide, Azathioprine, Cyclosporine A, Hydroxychloroquine, Methotrexate and Sulfasalazine, and two steroidal anti-inflammatory drugs, Prednisolone and Dexamethasone in this study. We examined the effects of these drugs on SRs (SR-A and CD36) and RCTs (ABCA1and ABCG1) expression, ox-LDL uptake, and total cholesterol content in THP-1-macrophage.
Our results showed that Leflunomide dose-dependently increased ABCA1 expression, which indicated Leflunomide might be able to enhance cholesterol efflux in macrophage. Meanwhile, we found SR-A expression, dii-oxLDL uptake as well as total cholesterol content were reduced in THP-1 macrophages after Cyclosporin A treatment. Moreover, HCQ increased both ABCA1 and ABCG1 protein expression. These findings suggested Leflunomide, Cyclosporine A and HCQ might modulate foam cell formation. Other tested DMARDs, including Azathioprine, Methotrexate and Sulfasalazine, had no apparent effect on SRs and RCTs levels. We further showed here that steroid drugs, including Prednisolone and Dexamethasone, reduced ABCA1 expression in THP-1-macrophage, which indicated that these two drugs might promote foam cell formation and increase risk of cardiovascular disease. Clinician should be aware of this when use these two drugs in the treatment of RA.
目次……………………………………………………………..……..….І
表目錄……………………………………………………………..……Ⅴ
圖目錄………………………………………………………………..…Ⅵ
中文摘要…………………………………………………….………….Ⅷ
Abstract…………………………………………………………......….Ⅹ
第一章 緒論
第一節 動脈粥狀硬化(atherosclerosis)……………..…………1
第二節 泡沫細胞的調控……………………………..………..2
第三節 類風溼性關節炎和動脈粥狀硬化之關係………..…..3
第四節 類風溼性關節炎的臨床用藥……………………...….4
第五節 研究目的與策略………………………………………4
第二章 材料與方法
第一節 細胞培養………….…………………..……………….6
第二節 藥品製備………………………………...…………….6
第三節 Oxidized low-density lipoprotein (ox-LDL)製備與測定………………………………………………………7
第四節 DiI-oxLDL製備…………………………….…..….....10
第五節 西方墨點法…………………………..……………….10
第六節 DiI-oxLDL吞噬能力測驗..……………………..……13
第七節 總膽固醇含量之測試…………...……………………14
第三章 結果
第一節 Leflunomide增加THP-1-macrophage 上ABCA1的表現量,而其不會影響DiI-oxLDL吞噬的能力以及總膽固醇之含量………………….……………………….16
第二節 Azathioprine不影響THP-1-macrophage上CD36、ABCA1和ABCG1之表現量,但有些微增加SR-A之趨勢,而其不影響THP-1-macrophage吞噬DiI-oxLDL的能力以及總膽固醇之含量………………………..17
第三節 Cyclosporine A會降低THP-1-macrophage上SR-A的表現量,以及會降低DiI-oxLDL吞噬的能力和總膽固醇之含量……………………………………….…….18
第四節 Hydroxychloroquine會增加THP-1-macrophage上ABCA1、ABCG1以及SR-A的表現量,並會增加DiI-oxLDL吞噬的能力但不會影響其總膽固醇之含量………………………………………………..……18
第五節 Methotrexate會些微增加THP-1-macrophage 上SR-A以及CD36之表現量、但不會影響DiI-oxLDL吞噬的能力以及總膽固醇之含量…………………………..19
第六節 Sulfasalazine不影響THP-1-macrophage SR-A、CD36、ABCA1和ABCG1蛋白表現量、DiI-oxLDL吞噬的能力以及總膽固醇之含量…………………..20
第七節 Prednisolone會降低THP-1-macrophage上ABCA1的表現量,但不會影響DiI-oxLDL吞噬的能力以及總膽固醇之含量………………………………………..…20
第八節 Dexamethasone會降低THP-1-macrophage上ABCA1的表現量,但不會影響DiI-oxLDL吞噬的能力以及總膽固醇之含量………………….…………………….21
第九節 Hydroxychloroquine影響THP-1-macrophage ABCA1 mRNA level………………………………………......21
第十節 Hydroxychloroquine影響THP-1-macrophage Sp-1以及AP-1的binding activity………………………………22
第十一節 Hydroxychloroquine增加THP-1-macrophage細胞核內Sp1之表現量…………………………………...22
第十二節 Hydroxychloroquine影響primary macrophage ABCA1以及ABCG1之表現量之機制……………...23
第四章 討論……………………………………………………………24
第五章 結論…………………………………………...……………….29
參考文獻………………………………………………………….…….30

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