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

感染性心內膜炎為細菌引起的心血管疾病。草綠色鏈球菌為主要致病菌，其中又以轉糖鏈球菌最常見，且基因已於2002年被完整定序，故本實驗室以轉糖鏈球菌研究心內膜炎的致病機轉。轉糖鏈球菌表面上的葡萄糖傳遞酶GTFC可以引起內皮細胞產生細胞激素IL-6。若以細胞骨架抑制劑cytochalasinD先與內皮細胞培養後，則會降低GTFC刺激內皮細胞產生IL-6。顯示GTFC須進入內皮細胞後才能引起發炎反應。此外，近年研究中指出，內皮細胞能產生較低濃度的活性氧分子造成發炎反應。因此我們假設GTFC必須經由內吞作用進入細胞，活化內皮細胞產生活性氧分子，分泌細胞激素。內吞作用包含Caveolae和Clathrin依賴型路徑，分別使用caveolae抑制劑MbetaCD以及Clathrin抑制劑MDC於內皮細胞刺激實驗中，發現MbetaCD不會使GTFC刺激的IL-6表現量下降，MDC則有部份抑制。螢光染色結果顯示GTFC不會與caveolin-1在同一位置；GTFC 與Clathrin則有重疊聚集的現象，故GTFC進入細胞的方式極有可能是藉由Clathrin依賴型路徑，而非經由caveolae的方式。GTFC和早期內小體標的蛋白EEA1有共同聚集現象，證實GTFC的確能進入內皮細胞內。活性氧分子的產生主要經由NADPH oxidase和eNOS的路徑，分別使用NADPH oxidase抑制劑apocynin及eNOS抑制劑L-NMMA，apocynin會使IL-6的產生下降，但L-NMMA則不會影響。以H2DCFDA螢光試劑偵測活性氧分子強度，證實GTFC會刺激內皮細胞產生活性氧分子，顯示GTFC利用內吞機制進入細胞，活化NADPH oxidase產生活性氧分子。參與GTFC活化內皮細胞產生活性氧分子的訊息路徑可待進一步探討。

關鍵字

轉糖鏈球菌；葡萄糖傳遞酶內皮細胞；內吞作用；活性氧分子

並列摘要

Infective endocarditis is a cardiovascular pathology that is often caused by bacteria.Viridans streptococci are major pathogens and Streptococcus mutans is the only member with its whole genomic sequence completed. Therefore, we focused on the investigation of virulence factors in S. mutans. We have identified previously that S. mutans, glucosyltransferase GTFC, induces endothelial cells activation to produce IL-6 and the activation could be blocked by pre-treatment of endothelial cells with cytochalasinD, a cellular skeleton inhibitor. This finding led us to hypothesize that GTFC may be internalized by endothelial cells to trigger an inflammatory responses through reactive oxygen species. Because, recent studies suggested that low concentrations of ROS in endothelial cells could induce inflammatory responses.Different inhibitors of endocytosis were tested to define whether caveolae- or clathrin-dependent pathway is associated with the internalization of GTFC. A clathrin-dependent inhibitor, MDC inhibited parally the production of IL-6, whereas the caveolae-related inhibitor, MβCD, failed to inhibit the GTFC induced cellular activation. Protein co-localization analysis by immunostaining indicated that GTFC could associate with clathrin but not caveolin-1, a finding correlated with that of inhibition analysis. GTFC could also co-localize with an early endosomal marker EEA1, further suggesting that clathrin-mediated endocytosis might be involved in GTFC internalization. Moreover, intracellular production of ROS, detected by H2DCFDA, was enhanced shortly after GTFC stimulation and ROS production could be inhibited by a NADPH oxidase inhibitor, apocynin, but not by an eNOS inhibitor, L-NMMA. Cellular signaling pathways involved in the GTFC-stimulated ROS production and subsequent activation were discussed.

並列關鍵字

S. mutans ； glucosyltransferase ； endothelial cells ； endocytosis ； ROS

參考文獻

吳淑惠. (2005). 篩選篩選體內表現基因載體的建立與心內膜炎免疫反應之特徵碩士國立臺灣大學醫學院口腔生物科學研究所碩士論文.

葉秋月 (2006). 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. 博士國立台灣大學醫學院微生物學研究所.

Ajdic, D., McShan, W. M., McLaughlin, R. E. & other authors (2002). Genome sequence of Streptococcus mutans UA159, a cariogenic dental pathogen. Proc Natl Acad Sci 99, 14434-14439.

Aoki, H., Shiroza, T., Hayakawa, M., Sato, S. & Kuramitsu, H. K. (1986). Cloning of a Streptococcus mutans glucosyltransferase gene coding for insoluble glucan synthesis. Infect Immun 53, 587-594.

Baddour, L. M., Christensen, G. D., Lowrance, J. H. & Simpson, W. A. (1989). Pathogenesis of experimental endocarditis. Rev Infect Dis 11, 452-263.

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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

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