2010-08-012024-05-13https://scholars.lib.ntu.edu.tw/handle/123456789/643031摘要：感染性心內膜炎是一種，高致死率，高復發率，不易治療的心血管感染疾病。草綠色鏈球菌是在心臟瓣膜不正常或受損的病人身上，最常被分離出引起亞急性感染性心內膜炎的伺機性致病菌。國外流行病學調查指出，最常自心內膜炎病患分離到的菌種為Streptococcus sanguinis、S. bovis、S. mutans、及S. mitis，而國內相關研究則指出轉糖鏈球菌(S. mutans)是草綠色鏈球菌中引發菌血症後，最容易導致心內膜炎的菌種，也是本實驗室長期研究的方向。我們長遠的目標是要研究感染性心內膜炎的致病機制，而這個計劃是著重於細菌引發心臟瓣膜及心內膜發炎的分子及免疫機制。由臨床感染性心內膜炎病患的組織病理切片，顯示草綠色鏈球菌在贅生物中形成群聚，並被血栓及纖維素緊密包裹，不會直接侵入心臟瓣膜或鄰近心內膜，因此細菌是如何引起心瓣膜持續性的發炎仍是未知，但卻是重要的臨床問題及研究主題。我們假設當細菌存在贅生物中，能持續釋出調節素刺激心瓣膜間質細胞產生細胞激素、及白血球趨化激素，引起單核細胞浸潤並維持發炎持續進行，細菌調節素是重要的致病因子，與心瓣膜間質細胞交互作用，可能參與幫助細菌引起持續性發炎反應的重要角色。我們之前的研究結果已證實，廣泛存在草綠色鏈球菌的葡萄糖傳遞酶(glucosyltransferase, GTF)是重要的調節素，可直接活化血管上皮細胞產生IL-6，在大鼠心內膜炎模型中也有相同作用，而GTF 也可在實驗性內膜炎之贅生物中被表現，這些初步結果證實了我們的假設，且本實驗室領先提出之調節素，在感染性心內膜炎致病機轉，扮演重要角色的概念已被認可並接受，即將發表在感染性疾病相關領域知名的期刊Jouranl of Infectious Diseases。在此次計畫中，我們將進一步探討GTF 活化人類心臟瓣膜間質細胞的分子機轉、及找到此細胞活化後釋出之趨化單核球及T 細胞的白血球趨化激素，並在實驗性心內膜炎的大鼠模型，及心內膜炎病患的組織病理切片確認其表現。並利用已建立之細胞模型評估使用專一性siRNA 抑制白血球趨化激素分泌之能力。這個計畫的主要目的是(1) 尋找並確認GTF 附著人類心臟瓣膜間質細胞的分子及活化的機轉。(2) 分析並鑑定瓣膜間質細胞趨化單核細胞及T 淋巴球的白血球趨化激素(3) 確認瓣膜間質產生之白血球趨化激素的功能及分析使用專一性siRNA 抑制之能力(4) 確認白血球趨化激素，在實驗性心內膜炎的大鼠模型、及心內膜炎病患的組織病理切片之表現這個計畫的優勢及前驅性，在於我們已建立人類心臟瓣膜間質細胞培養技術，並可利用已建立好的實驗性心內膜炎的大鼠模型，來檢測這些致病因子的重要性。本計畫的協同研究人員將包括臨床病理醫師，協助病患的組織病理切片染色。這個計畫的重要性，是對瞭解人類心臟瓣膜間質細胞，在感染症所引發之發炎反應中是扮演重要角色，並找出與心臟內膜發炎相關的白血球趨化激素，對治療心內膜發炎將有重大貢獻。確認具專一性抑制能力之siRNA 將申請專利。<br> Abstract: Infective endocarditis (IE) is an infection disease of the cardiovascular system, andcarries a high recurrence and morality rate. Viridans streptococci are the most commonpathogens of IE in patients with abnormal or injured heart valves. The most commonstreptococci isolated from patients with endocarditis are Streptococcus sanguinis, S. bovis, S.mutans, and S. mitis. In Taiwan, S. mutans is responsible for the highest incidence ofendocarditis in bacteremia-associated pyogenic infections. Our broad and long-term objectiveis to investigate the pathogenesis of viridans streptococci induced IE, and this proposal willfocus on the interaction of human cardiac valve interstitial cells (VICs) with common viridansstreptococcal modulin, glucosyltransferease (GTF). Histopathologically, the pathogenesis ofIE is characterized by the formation of septic vegetation (fibrin-platelet matrix with bacterialclusters embedded inside) and inflammatory reaction in infected valves or adjacentendocardium. The molecular mechanism of the persistent inflammatory responses during IEremains unclear. We hypothesized that streptococcal modulins, secreted in cell-free form,might easily gain egress from the embedded vegetations into the surrounding valvular stroma,where the interaction with VICs would take place directly to recruit inflammatory infiltrates.We demonstrated previously that streptococcal modulins, GTF, in either cell-bound or freeforms, could activate directly endothelial cells to recruit monocytes in vitro and couldmodulate inflammatory cytokine production in situ in an experimental endocarditis rat model.Furthermore, we demonstrated recently that S. mutans embedded inside fibrin-platelet clotsare active in secreting GTFs and VICs at infected valve could be activated in experimental orhuman IE. These results provide direct supports to confirm our hypothesis and our leadingfindings in the importance of modulin in the pathogenesis of IE has been well-recognized andaccepted to be published in the leading Journal of this field “Journal of Infectious Diseases”.In this continued study, we proposed to investigate further the molecular interaction of S.mutans GTF with human VICs and to delineate the mediators that direct chemotaxis ofmonocytes and T cells. In addition, the potential of using siRNA for inhibition of thesechemokines will also be investigated.The specific aims of this three-year project are:1. Identify specific pathogen recognition receptors on VICs and signaling pathwaysinduced by GTFs.2. Identify chemokines involved in chemotaxis of monocytes, CD4+ or CD8+ T cells byactivated VICs using chemokine RT-PCR arrays.3. Confirm and characterize functions of chemokines involved in chemotaxis by trans-wellmigration and the potential of specific siRNA inhibition.4. Confirm expression of chemokines in vivo in a rat experimental or human endocarditis.The significance of this pioneering proposal is to identify molecular mechanisms andmediators involved in endocardiac inflammatory response against infectious agent. Resultsobtained will have clinical implications to the understanding of pathogenesis of IE. Thisproposal will include collaboration with sophisticated clinical pathologist as well aswell-trained laboratory personnel for cell culture. Specifically, the common or uniquechemokines released by VICs in recruiting leukocytes and inducing inflammatory responsewill be investigated initially using in vitro cell culture systems and confirmed subsequentlyin vivo by using experimental and human endocarditis. In addition, the potential of siRNAinhibition for the selected chemokines of interest will also be evaluated and submitted forpatent application.感染性心內膜炎草綠色鏈球菌調節素發炎反應白血球趨化激素干擾 性核醣核酸infective endocarditisviridans streptococcimodulininflammationleukocyte chemotaxissiRNA