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  • 學位論文

草綠色鏈球菌葡萄糖傳遞酶與內皮細胞的交互作用參與感染性心內膜炎的免疫致病機制

Viridans streptococcal glucosyltransferases–endothelial cell interactions in the immunopathogenesis of infective endocarditis

指導教授 : 賈景山

摘要


感染性心內膜炎,是細菌感染心臟瓣膜所引起的疾病,主要病徵是在心臟瓣膜上形成贅生物,造成慢性且持續性發炎反應。本論文主要是探討葡萄糖傳遞酶在感染性心內膜炎致病機轉上所扮演的角色。葡萄糖傳遞酶表現在多種會引起心內膜炎的草綠色鏈球菌的菌表面上,或是被細菌分泌出來,功能是代謝分解蔗糖並合成葡聚糖,葡聚糖引起細菌聚集和生物膜形成,是導致齲齒的重要致病因子。另一方面,葡萄糖傳遞酶也是細菌性調節素,刺激人類和老鼠單核球產生大量IL-6。在人類臍帶靜脈內皮細胞中,不管是在細菌、細菌萃取物中、或是純化的重組性葡萄糖傳遞酶,都可以專一性透過NF-kB活化,產生IL-6和黏附分子,黏附分子的表現,會促使人類單核球細胞株U937黏附到活化內皮細胞上,加入純化的IL-6和IL-6Rα會增強U937的黏附現象。更進一步的研究顯示葡萄糖傳遞酶,可在心內膜炎動物模式體內表現,而且與心臟瓣膜發炎反應急性期IL-6產生相關。草綠色鏈球菌感染的心內膜炎病人心臟瓣膜切片中,在心臟瓣膜、新生血管的內皮細胞、浸潤的單核球、和心臟纖維母細胞有細胞激素表現。體外培養的心臟纖維母細胞,會被葡萄糖傳遞酶刺激單核球所產生的IL-1β和TNF-α刺激產生MCP-1。綜合以上發現,我們提出一個感染性心內膜炎的發炎機制,當感染性心內膜炎發炎反應產生時,細菌性調節素葡萄糖傳遞酶會活化內皮細胞,引起單核球趨化和浸潤。再者,經由葡萄糖傳遞酶活化浸潤單核球所分泌的細胞激素,活化心臟纖維母細胞,產生趨化激素促使單核球趨化,導致慢性且持續性發炎反應。艱難梭狀桿菌毒素A羧基端(ARU)與葡萄糖傳遞酶有很高的相似性,最近研究發現ARU具有免疫佐劑功能。第二部份結果顯示rARU刺激內皮細胞產生IL-6、IL-8和MCP-1,需要胎牛血清和鈣離子的參與。抑制劑結果顯示rARU是透過PI3K、PKC、PTK、MAPKs和NF-kB的活化產生細胞激素,IL-8和MCP-1的產生會趨化單核球和多形核球,表示rARU會引起局部發炎反應產生,促使不容易產生免疫反應的抗原,被趨化前來的吞噬細胞辨認和呈現,而局部產生的IL-6促進B淋巴球分化成為漿細胞,產生專一性的抗體。綜合以上兩部份的研究,我們推論葡萄糖傳遞酶的羧基端與ARU相似的序列,可能是負責與內皮細胞結合的區域。

並列摘要


Infective endocarditis (IE), a microbial infection in the heart valves, is characterized by vegetation formation and chronic endocardial inflammation. In the present study, we examined the role of glucosyltransferases (GTFs) found in viridans streptococci-induced infective endocarditis. GTFs were discovered in several IE-inducing streptococcal species, and presented as either the bacterial surface or extracellular enzyme, to synthesize glucan that cause bacterial aggregation and biofilm formation on tooth surfaces. GTFs were also identified to be modulins of IL-6 production in monocytes of human or rat. When treated on HUVEC, both bacteria-bound and -free GTFs could induce IL-6 and adhesion molecule expression through NF-kB activation. Activated endothelial cells enhance adhesion of monocytes and such adhesion can also be achieved by treating the cells with a mixture containing IL-6 and IL-6Rα. Furthermore, we demonstrate that GTF is detected in situ and induces IL-6 synthesis during acute phase of S. mutans-induced rat experimental endocarditis. In clinical specimens from viridans streptococci induced endocarditis, secretion of pro-inflammatory cytokines was detected on valvular, neocapillary endothelial cells, infiltrated macrophages and cardiac fibroblasts. Cardiac fibroblasts cultured in vitro can be activated synergistically by IL-1β and TNF-α, released from GTF-activated monocytes, to release MCP-1. Taken together, we propose a mechanism for endocardial inflammation during IE for recruitment and retention of monocytes, the former may be activated by bacterial modulins on endothelial lining, while, the latter, may be maintained by cardiac fibroblasts through cytokines and chemokines. The C-terminal repetitive domain of Clostridium difficile toxin A (ARU) is homologous to GTF and exhibites adjuvant activity in mucosal immunity. In second part of this study, we demonstrate that ARU is sufficient to induce IL-6, IL-8 and MCP-1 production in endothelial cells through serum- and calcium-dependent manner. Inhibition results demonstrate that cytokine production in ARU-activated HUVEC is through PI3K、PKC、PTK、MAPKs and NF-kB activation. Both IL-8 and MCP-1 trigger leukocytes chemotaxis in vitro. These results suggest that mucosal adjuvant activity of C-terminus of TcdA may also attribute to the interaction of endothelial cells to release IL-6, which is important for maturating plasma cells. Take together; we hypothesized that the C-terminal repeates of GTFs, similar to ARU, might be involved in the binding of endothelial cells.

參考文獻


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被引用紀錄


陳詩薏(2007)。轉糖鏈球菌葡萄糖傳遞酶活化人類臍帶靜脈內皮細胞之機制〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2007.02755
張家榮(2007)。人類心臟瓣膜纖維母細胞受草綠色鏈球菌葡萄糖傳遞酶活化引發白血球趨化作用之研究〔碩士論文,國立臺灣大學〕。華藝線上圖書館。https://doi.org/10.6342%2fNTU.2007.01579

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