臺灣博碩士論文加值系統

過去研究發現，修飾型低密度脂蛋白(modified-low-density lipoprotein)如:氧化型低密度脂蛋白 (oxidized low-density lipoprotein, oxLDL)或糖化型低密度脂蛋白提高時,會增加動脈粥狀硬化的發生率，而巨噬泡沫細胞與動脈粥狀硬化的發展息息相關。巨噬泡沫細胞形成的原因，主要為巨噬細胞透過其細胞膜表面清道夫受體 (Scavenger Receptors, SRs)吞噬修飾型LDL，與逆向轉運載體蛋白家族 (ATP-binding cassette transporters, ABC transporters)排出膽固醇的調控失衡，使膽固醇累積於巨噬細胞所致。相關研究指出，巨噬泡沫細胞大量表現了類鐸受體-2及-4型(Toll-like Receptor 2 and 4, TLR-2 and -4)，且TLRs受到諸多配體(ligand)的影響，如：lipopolysaccharide (LPS)或修飾型LDL，進而促使巨噬泡沫細胞發展及提高發炎因子產生；此外，亦有研究指出修飾型LDL可透過TLR-4提高巨噬細胞產生活性氧化物，進而增加發炎因子表現。故修飾型LDL與TLRs之間的交互作用，與動脈粥狀硬化發展是有相關性的。
美弗明(Metformin)為目前臨床治療第二型糖尿病的第一線用藥，治療史已長達五十多年。Metformin除改善高血脂外，亦可使人類血管內皮細胞減少Vascular Cell Adhesion Molecule-1 (VCAM-1)的表現，使單核球不易附著在血管內皮細胞表面，進而減少內皮細胞下空隙巨噬細胞的累積，減緩巨噬泡沫細胞的發展；此外，Metformin亦透過活化腺核苷單磷酸活化蛋白激酶(AMP-activated protein kinase, AMPK)，降低內皮細胞因Interleukin-1-β所誘發發炎因子的表現。
本研究以J774A.1 murine macrophage為模型，給予oxLDL誘使巨噬細胞形成巨噬泡沫細胞，並觀察Metformin對於oxLDL所誘發的巨噬泡沫細胞發展，以及發炎因子表現之可能機制。結果顯示J774A.1巨噬細胞中油滴的累積，隨著oxLDL (50μg/ml)處理的時間增加而上升，並在處理的24小時內，第18小時油滴累積量有較多的趨勢。在TLRs及發炎因子方面，oxLDL處理0, 2, 4, 8, 12, 18, 24小時，可以提高TLR-2及-4的基因表現，且TLR-2於18小時後達顯著差異，然而TLR-4於2小時後即有顯著性差異，並持續至24小時。在發炎指標MCP-1、IL-6及TNF的部份，oxLDL處理12-24小時可顯著提高前述因子的基因表現。單獨處理Metformin (10-9, 10-7, 10-5, 10-3 M) 18小時，結果顯示隨著Metformin濃度提高，TLR-2、-4及發炎指標MCP-1, IL-6及TNF的基因表現逐漸下降。而oxLDL與Metformin (10-5 and 10-3 M) 共同處理18小時的實驗中，發現Metformin可有效改善oxLDL所造成的油滴累積，並顯著抑制oxLDL所誘發的TLR-2及-4基因表現。此外，Metformin亦可改善oxLDL所誘發的MCP-1、IL-6以及TNFα基因表現。進一步探討Metformin對巨噬泡沫細胞發展以及發炎因子調控的機制，使用p-AMPK抑制劑compound C ( 20 M) 預先處理J774A.1巨噬細胞 1小時後，再以Metformin (10-3M) 或AICAR (10-3M) 處理1小時，結果顯示AICAR與Metformin可提高AMPK磷酸化，而compound C可抑制此結果。並且，oxLDL (50 g/ml)及Metformin (10-3 M)合併處理compound C 18小時的實驗中，發現Metformin改善oxLDL所提高之TLR-4、MCP-1及TNFα基因表現，可以透過AMPK的磷酸化路徑來達成；然而，Metformin對於oxLDL所提高之TLR-2及IL-6基因表現，並非透過AMPK磷酸化路徑，其詳細機制仍須進一步地探討。

Modified-low density lipoproteins (LDL), like oxidized-LDL or Glycated-LDL have been shown to associate with the risk of atherosclerotic cardiovascular disease. In addition, ox-LDL is responded to macrophage foam cell formation. Cholesterol homeostasis of macrophage is tightly regulated by scavenger receptor and ATP-binding cassette (ABC) transporters during the transformation of macrophage foam cells. Moreover, recent evidences reveal the involvement of Toll-like receptors in macrophage foam cell formation. Macrophage-derived foam cell is clarified to induce inflammation and plays a critical role in the initiation and progression of atherosclerosis.
Metformin has been used over fifty years, which is used as first-line drug of choice for the treatment of type 2 diabetes. Reports indicate that Metformin ameliorates Hyperlipidemia in human and reduced vascular cell adhesion molecule expression in human endothelial cells. Although the underlying mechanisms of Metformin reducing macrophages foam cell formation are not fully understood, some researchers believe AMP-activated protein kinase, AMPK, is the candidate.
In our condition, J774A.1 murine macrophage was the model we used. We induced macrophage foam cell formation by ox-LDL and determined macrophages lipid accumulation by Oil red O stating. Results gave reproducible evidence that ox-LDL increased macrophages lipid accumulation after 18 hours. Besides, TLR gene (TLR-2 and 4) and inflammatory gene (MCP-1, IL-6, and TNFwere significantly induced under the same condtion. Interestingly, TLR-4 was induced earlier than TLR-2. According to the results of ox-LDL induced macrophage in presence or absence of Metformin, we found it was Metformin that decreased ox-LDL-induced macrophage foam cell formation and inflammation. Also, Metformin had the effect of decreasing macrophage basal inflammatory gene and TLR gene in our condition. To understand the role of AMP-activated protein kinase (AMPK) in this experiment, compound C, the AMPK antagonist, was used to investigate whether the effect of Metformin on ox-LDL-induced macrophage foam cell through AMPK pathway. We found Metformin attenuated oxLDL-induced TLR-4、MCP-1and TNFα through AMPK activation However, TLR-2 and IL-6 were not regulated by AMPK activation.
We tried to figure out the possible role of Metformin in ox-LDL-induced macrophage foam cell formation. After investigating the involvement AMPK in this condition, we found Metformin on foam cell formation and its function was through AMPK pathway. We believed Metformin had benefit to solve foam-cell-related physiologic problems.

目錄

中文摘要 ------------------------------------------------------------------------
i
英文摘要 ------------------------------------------------------------------------
iii
目錄 ------------------------------------------------------------------------------
v

表目錄與圖目錄 ---------------------------------------------------------------
vii

中英文名詞對照表及英文縮寫 ---------------------------------------------
ix
第壹章
第壹章
第壹章
第壹章
第壹章 文獻回顧

一、 動脈粥狀硬化(Atherosclerosis) 1

二、 巨噬細胞生理功能 2
三、 脂蛋白與巨噬細胞膽固醇恆定 3
四、 低密度氧化脂蛋白 (Oxidized Low Density Lipoprotein;
oxLDL) 6
五、 oxLDL與動脈粥狀硬化之關係 7
六、 類鐸受體 (Toll-like Receptors; TLRs) 8
七、 TLRs與oxLDL與動脈粥狀硬化之關係 8
八、 美弗明(Metformin)
9
九、 Metformin與動脈粥狀硬化之關係
9
十、 研究假說與研究目標 10
第貳章 材料與實驗方法
一、 藥品與材料 12
二、 抗體 13
三、 細胞培養 13
四、 低密度脂蛋白氧化 (oxidized-LDL) 13
五、 TBARS測定 14
六、 Oil-red-O 染色與定量 14
七、 細胞全蛋白萃取 15
八、 西方墨點法(Western Blot) 15
九、 RNA 樣本製備 16
十、 反轉錄 (Reverse Transcription, RT) 16
十一、 聚合酶連鎖反應 (Polymerase Chain Reaction, PCR) 17
十二、 統計分析 17
第參章 實驗結果
一、
一、
一、 氧化低密度脂蛋白對於巨噬細胞內脂質累積和泡沫化影
響 18
二、 氧化低密度脂蛋白對於類鐸受體-2及-4與發炎因子MCP-1、IL-6及TNF 基因表達之影響
18
三、 Metformin對於類鐸受體-2及-4與發炎因子MCP-1、IL-6及TNF 基因表達之影響
18
四、 Metformin對於氧化低密度脂蛋白誘導之類鐸受體-2及-4與發炎因子MCP-1、IL-6及TNF基因表達之影響 19
五、 AMPK在Metformin調控氧化低密度脂蛋白誘導之巨噬泡沫細胞發展以及發炎因子表現之影響 19
第肆章 結論
21
參考文獻
25
表
31
圖
32
附錄 43

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