類鐸受體於粥狀動脈硬化之角色及其於缺血性腦中風之訊息傳遞路徑研究

Exploring the Role of Toll-like Receptors on Atherosclerosis and its Signaling Pathway in Ischemic Stroke

鄔定宇(Dean Wu)

指導教授 ： 胡朝榮

共同指導教授 ： 許重義

臺北醫學大學/醫學院/臨床醫學研究所/博士(2014年)

https://doi.org/10.6831/TMU.2014.00058

摘要

粥狀動脈硬化是一種慢性發炎性的疾病，其中內皮細胞與單核球都有參與這個過程。單核球與內皮細胞的附著需要靠附著因子的表現，例如血管細胞附著分子1。類鐸受體4已經在粥狀硬化斑塊中有表現，而這樣的血管硬化過程中附著分子的表現增加是經由NF-kB的調控的。 在第一個實驗中，我們想要了解類鐸受體4對於內皮細胞附著分子表現的影響以及類鐸受體4對單核球與內皮細胞附著的影響。我們發現不論在mRNA或者是蛋白質，LPS可以導致內皮細胞血管附著分子1的表現增加，而這樣的表現增加卻可以被抗類鐸受體4的抗體所減弱。同時單核球與內皮細胞的附著可以因LPS的促發而增加，但是這個附著的增加也可以被抗類鐸受體4與抗血管細胞附著分子1的抗體所減弱。因此我們下了一個結論就是類鐸受體4的配體LPS，經由類鐸受體4使得內皮細胞的血管細胞附著分子1表現增加，而且會影響到單核球與內皮細胞的附著，因此我們認為類鐸受體4及其下游路徑可能對於粥狀動脈硬化扮演了重要的角色。
過去的研究證據顯示類鐸受體參與了動脈硬化與缺血性腦中風的過程，但是缺血性腦中風患者的類鐸受體基因的表現卻從未被研究過，在我們的第二個實驗中，我們測量了缺血性腦中風患者周邊血液的類鐸受體2, 3, 4 mRNA相對於控制組的表現情形。結果我們發現缺血性腦中風患者的周邊血液中類鐸受體4的mRNA表現有增加，但是類鐸受體2, 3的mRNA則無增加的現象。我們同時也去測量IL8這個與動脈硬化有關且又是類鐸受體4下游分子的基因表現，結果發現IL8的mRNA表現是下降的。進一步分析起來，我們所有的缺血性腦中風患者都有使用阿斯匹靈作為缺血性腦中風的次級預防，而阿斯匹靈在過去就被證實可以抑制NFkB，而NFkB又可以調控IL-8的表現增加。因此我們推測缺血性腦中風因為類鐸受體4表現增加所引發的發炎反應是可以部份的被阿斯匹靈所抑制。
根據我們第二個研究的結果顯示類鐸受體4在缺血性腦中風患者的表現是增加的，然而，缺血性腦中風患者類鐸受體4下游的路徑在過去並未被研究過，因此，在我們第三個實驗中，我們從第二個研究中的疾病組與控制組中各別選取了類鐸受體4表現最高的12位個案，分別測試類鐸受體4下游路徑的基因在兩組中是否出現差異，並且我們使用這些有表現差異的基因來做下游路徑的分析，並進一步藉由KEGG Mapper來視覺化這個路徑。我們發現缺血性腦中風的患者其類鐸受體4的下游路徑走的是非MyD88的路徑，UBE2V1-TRAF6以及TRAF6可能是缺血性腦中風最重要的基因表現路徑與下游目標分子。我們的結論是，通過我們使用的研究策略，我們更進一步的證實了缺血性腦中風患者類鐸受體4的下游路徑，也為未來的研究或臨床治療找到了可以介入的目標分子。

關鍵字

類鐸受體 ； 腦中風 ； 粥狀動脈硬化

並列摘要

Atherosclerosis is a chronic inflammatory disease in which both endothelial cells and monocytes are involved. Monocyte-endothelium adhesion is heavily dependent on the expression of adhesion molecules, such as vascular cell adhesion molecule 1 (VCAM-1). Toll-like receptor 4 (TLR4) has been found in atheroma and contributes to atherogenesis via the nuclear factor-kappa B signal pathway, a process within which adhesion molecules are regulated. In the first study, we explore the role of TLR4 in the adhesion molecule expression in endothelial cells and monocyte-endothelium adhesion. The anti-TLR4 antibody could suppress the overexpression of VCAM-1 induced by lipopolysaccharide (LPS) in messenger RNA and protein levels. The monocyte-endothelium adhesion triggered by LPS on human coronary artery endothelial cells was tested by anti-TLR4 antibody and anti-VCAM-1 antibody. Both anti-TLR4 antibody and anti-VCAM-1 antibody interfere with monocyte-endothelium adhesion. We conclude that LPS, a ligand of TLR4, upregulates VCAM-1 by interacting with TLR4 and then enhances monocyte-endothelium adhesion. TLR4 downstream signaling pathway might play an important role in atherogenesis.
Previous evidences support that Toll-like receptors are involved in the atherosclerosis and ischemic stroke. However, the expressions of TLRs genes in mRNA level in stroke patients have never been studied before. In our second study, we measured the mRNA levels of TLR2, TLR3, and TLR4 in the peripheral blood among stroke patients and non-stroke controls. We found TLR4 but not TLR2 nor TLR3 mRNA levels increased in the stroke patients. We further examined IL8 which is an atherosclerosis-associated mediator in TLR4 down-stream signaling pathway. Contradictorily, IL-8 mRNA levels significantly decreased in the patient group. Because all the patients were taking aspirin for secondary prevention, and aspirin has been proved to be an inhibitor of nuclear factor-kappa B, which upregulates IL-8 expression. Therefore, it is speculated that inflammation induced by over-expression of TLR4 could be suppressed, at least partially, by aspirin treatment.
Based on our second study, the expression of TLR4 is up-regulated in ischemic stroke, at least in subacute stage. However, the TLR downstream pathways in the context of stroke are not well studied before. In our third study, 12 ischemic stroke patients and 12 controls that had highest levels of TLR4 mRNA in the peripheral blood were selected from each group for further study. The differences of TLR downstream signaling pathways were analyzed and further visualized based on the TLR pathways in Kyoto Encyclopedia of Genes and Genomes (KEGG). We found that the genes up-regulated in stroke patients are involved in the MyD88-independent pathway and UBE2V1-TRAF6 ubiquitin-mediated proteolysis and TRAF6 could be the most important molecules among TLR downstream pathway in ischemic stroke. In conclusion, based on this strategy, we identified the more specific signaling pathways for ischemic stroke and provided few potential candidate molecules for further researches and clinical application.

並列關鍵字

TLR ； stroke ； atherosclerosis

參考文獻

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