賈景山臺灣大學：口腔生物科學研究所陳詩薏Chen, Shih-YiShih-YiChen2007-11-262018-07-092007-11-262018-07-092007http://ntur.lib.ntu.edu.tw//handle/246246/51314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。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.中文摘要---------------------------------------------------------------------------------------------I 英文摘要--------------------------------------------------------------------------------------------II 目錄-------------------------------------------------------------------------------------------------III 圖目錄----------------------------------------------------------------------------------------------VI 附圖目錄------------------------------------------------------------------------------------------VII 第一章 緒論-----------------------------------------------------------------------------------------1 一、人類齟齒致病菌—轉糖鏈球菌(Streptococcus mutans)------------------------------1 二、轉糖鏈球菌造成齟齒之致病機轉-------------------------------------------------------2 三、感染性心內膜炎(Infective Endocarditis, IE)的致病機轉-----------------------------3 1. 定義與分類--------------------------------------------------------------------------3 2. 牙科治療(Dental procedure)與感染性心內膜炎之關係---------------------4 3. 致病機轉(Pathogenesis) ----------------------------------------------------------5 四、轉糖鏈球菌之主要致病因子－葡萄糖傳遞酶----------------------------------------7 五、宿主的防禦機制(Host Defences)--------------------------------------------------------9 六、細菌－宿主的交互關係-----------------------------------------------------------------10 1. 細胞表面的黏附因子------------------------------------------------------------10 2. 細菌黏附造成宿主細胞產生細胞激素---------------------------------------12 3. 細菌內吞作用(Internalization)--------------------------------------------------12 七、活性氧分子(Reactive Oxygen Species, ROS)的產生-------------------------------16 第二章 實驗方法與材料------------------------------------------------------------------------18 1. 葡萄糖傳遞酶(Glucosyltransferase, GTFC)蛋白純化----------------------18 2. 蛋白質濃度測定------------------------------------------------------------------19 3. 人類臍帶靜脈內皮細胞(Human umbilical vein endothelial cell, HUVEC)分離方法--------------------------------------------------------------------------------20 4. 流式細胞儀(Flow Cytometry)分析人類臍帶內皮細胞分離之純度-----21 5. 聚丙烯胺膠體電泳法(SDS-PAGE)--------------------------------------------21 6. 西方點墨法(Western blot) ------------------------------------------------------22 7. 銀染色法(Silver stain) -----------------------------------------------------------22 8. 細胞刺激方法---------------------------------------------------------------------23 9. 酵素連結免疫吸附法(Enzyme linked immuno-absorbed assay, ELISA)23 10. 細胞螢光染色及顯微鏡觀察---------------------------------------------------24 11. 細胞質核分離---------------------------------------------------------------------25 12. 活性氧分子(Reactive Oxygen species, ROS)測定---------------------------25 13. 核糖核酸(RNA)萃取-------------------------------------------------------------26 14. 反轉錄聚合酶連鎖反應(RT-PCR)與聚合酶連鎖反應(PCR)-------------27 第三章 結果---------------------------------------------------------------------------------------29 一、重組蛋白質葡萄糖傳遞酶的純化與人類臍帶靜脈內皮細胞的分析------------29 二、葡萄糖傳遞酶黏附到內皮細胞的能力------------------------------------------------29 三、葡萄糖傳遞酶影響內皮細胞的骨架排列---------------------------------------------30 四、 偵測葡萄糖傳遞酶在內皮細胞中的位置--------------------------------------------31 五、葡萄糖傳遞酶進入細胞的內吞路徑-----------------------------------------------------32 六、葡萄糖傳遞酶刺激內皮細胞產生活性氧分子-----------------------------------------33 第四章 討論---------------------------------------------------------------------------------------36 一、葡萄糖傳遞酶能進入內皮細胞內活化產生IL-6-------------------------------------36 二、葡萄糖傳遞酶趨向以Clathrin依賴型內吞路徑進入細胞--------------------------38 三、葡萄糖傳遞酶可刺激內皮細胞活化產生活性氧分子------------------------------40 第五章 參考文獻---------------------------------------------------------------------------------42 圖目錄 圖一、重組蛋白質葡萄糖傳遞酶純化與人類臍帶靜脈內皮細胞之分析--------------53 圖二、葡萄糖傳遞酶對內皮細胞的黏附作用-----------------------------------------------54 圖三、抑制劑處理對葡萄糖傳遞酶刺激內皮細胞產生IL-6的影響---------------------55 圖四、葡萄糖傳遞酶影響內皮細胞之細胞骨架的排列-----------------------------------56 圖五、偵測葡萄糖傳遞酶在內皮細胞中的位置--------------------------------------------57 圖六、Caveolae標的蛋白caveolin-1之免疫螢光染色分析--------------------------------58 圖七、葡萄糖傳遞酶進入細胞的內吞路徑--------------------------------------------------59 圖八、早期內小體標的蛋白EEA1免疫螢光染色分析-------------------------------------60 圖九、 內吞作用抑制劑對葡萄糖傳遞酶刺激內皮細胞產生細胞激素的影響--------61 圖十、活性氧分子阻斷劑對葡萄糖傳遞酶活化內皮細胞的影響-----------------------62 圖十一、螢光及時影像偵測內皮細胞中活性氧分子的產生-----------------------------63 圖十二、葡萄糖傳遞酶刺激內皮細胞產生活性氧分子之強度--------------------------64 圖十三、抑制劑對葡萄糖傳遞酶刺激內皮細胞產生活性氧分子之結果--------------65 附圖目錄 附圖一、細胞的內吞作用包含吞噬作用(phagocytosis)和胞飲作用(pinocytosis) ----66 附圖二、Clathrin依賴型內吞路徑--------------------------------------------------------------67 附圖三、Caveolin依賴型內吞路徑------------------------------------------------------------68 附圖四、細菌經由宿主細胞的內吞機制進入細胞-----------------------------------------69 附圖五、血小板生長因子(platelet-derived growth factor, PDGF)誘導上皮細胞的活性氧分子生成----------------------------------------------------------------------------------------70 附圖六、內小體中的NADPH oxidase引起活性氧分子生成------------------------------716226506 bytesapplication/pdfen-US轉糖鏈球菌葡萄糖傳遞酶內皮細胞內吞作用活性氧分子S. mutansglucosyltransferaseendothelial cellsendocytosisROSotherhttp://ntur.lib.ntu.edu.tw/bitstream/246246/51314/1/ntu-96-R94450016-1.pdf