臺灣博碩士論文加值系統

革蘭氏陽性菌是引發感染性心內膜炎的常見致病菌，而目前認為循環中的血小板在感染性心內膜炎的病理上扮演重要角色。血小板會幫助細菌黏附於心內膜上而引發感染，接著細菌會再使更多的血小板聚集在感染的區域而形成更大的凝集塊，當凝集塊掉落隨著血液循環流到身體各處，可能發展成全身性血栓。細菌與血小板之間的作用在細菌引發心內膜炎上是一個重要的致病機轉，因此，若能了解細菌與血小板之間的作用模式，將有助於發展出更有效果的治療策略。
在本實驗室之前的研究顯示，纖維蛋白原(fibrinogen)在金黃色葡萄球菌(Staphylococcus aureus)與血小板的作用上扮演了重要的角色，金黃色葡萄球菌可藉由以血漿中的纖維蛋白原當做橋樑來與血小板作用進而引發血小板的活化凝集。化膿性鏈球菌(Streptococcus pyogenes)是臨床上一種重要的致病菌，已證明可以引發血小板的活化凝集，雖然有許多研究致力於化膿性鏈球菌與血小板之間的作用探討，但化膿性鏈球菌引發血小板活化凝集的詳細機轉目前仍無定論。在本實驗中，我們將探討化膿性鏈球菌引發血小板活化凝集的機轉，以期進一步解開化膿性鏈球菌與血小板之間作用的謎底。
從實驗結果發現，化膿性鏈球菌的臨床菌株M29588及78M6於富含血小板血漿中在延遲幾分鐘後會引發血小板的活化進而凝集，而化膿性鏈球菌引發血小板活化凝集的活性會被血小板受體GP IIb/IIIa的抑制劑tirofiban (Aggrastat®)以及可以藉由與纖維蛋白原結合而阻斷纖維蛋白原媒介的血小板凝集作用的重組蛋白ClfA221-550給抑制掉。化膿性鏈球菌引發血小板活化凝集的活性與金黃色葡萄球菌十分相似。我們也發現了金黃色葡萄球菌在血小板懸浮液外加纖維蛋白原的情況下能夠引發血小板的活化凝集，但是化膿性鏈球菌即使在纖維蛋白原存在下仍然無法引起血小板活化凝集。結果顯示，雖然纖維蛋白原在化膿性鏈球菌與血小板的作用上扮演了不可缺少的角色，其他血漿因子在化膿性鏈球菌引發血小板活化進而凝集的過程是必需的。
在本研究中，我們也利用重組蛋白ClfA221-550製成親和性膠體管柱，藉由其可與纖維蛋白原結合的特性來純化血漿中的纖維蛋白原。從SDS-PAGE電泳結果可觀察到高純度的纖維蛋白原，而功能測試的結果證明純化的纖維蛋白原具有幫助血小板吸附、凝集的作用，並且在凝血酵素的作用下會形成纖維蛋白凝集塊。

Circulating platelets play an important role in the pathogenesis of infectious endocarditis, a severe disease caused mostly by the infection of Gram-positive bacteria. Platelets may help bacteria attach to endocardium, initiating endocarial infection, followed by facilitating the formation of macroscopic vegetation which may disperse away from valves, resulting in systemic embolization. Given the interaction of bacteria with platelets is a critical virulent mechanism for bacteria to induce infectious endocarditis, a better understanding of the processes involved in bacterium-platelet interaction would be meaningful in the design of more efficacious therapeutic strategy for the prevention and treatment of infectious endocarditis.
Our previous study revealed that plasma fibrinogen mediates Staphylococcus aureus (S. aureus)-platelet interaction by acting as a bridging molecule, leading to the activation and aggregation of platelets. Streptococcus pyogenes (S. pyogenes) is one of the clinically important pathogens that can cause platelet activation and aggregation. Although a variety of studies have been devoted to elucidate the mechanism underlying S. pyogenes-platelet interaction, the precise mechanism by which S. pyogenes triggers platelet activation and aggregation remains controversial. In order to unravel the mystery regarding the S. pyogenes-platelet interaction, this study was undertaken to elucidate the mechanism underlying the response of platelets to S. pyogenes.
It was found that both S. pyogenes M29588 and S. pyogenes 78M6 caused platelet activation and aggregation in platelet-rich plasma with a lag of several minutes and their platelet reactivity could be abrogated either by the platelet GP IIb/IIIa antagonist tirofiban (Aggrastat®) or ClfA221-550, which binds to the γ chain C-terminus of fibrinogen, leading to the inhibition of fibrinogen-mediated platelets aggregation. This platelet reactivity of S. pyogenes appears to be similar to that of S. aureus 30326. We also found that fibrinogen supplement enables S. aureus 30326 to induce activation and aggregation of washed human platelets, which was not observed with S. pyogenes bacteria. All the results indicate that although fibrinogen plays an indispensable role in causing aggregation of platelets by S. pyogenes bacteria, some other plasma factor(s) might be also required for S. pyogenes bacteria to induce platelet activation and the subsequent aggregation.
In this study, we also develop a convenient method for the purification of plasma fibrinogen using a ClfA221-550-based affinity column. Highly purified fibrinogen was obtained with this method as examined by SDS-PAGE and it was shown able to support platelet adhesion, aggregation and form fibrin clot in response to thrombin.

1.縮寫表------------------------------------------------------------------------------2
2.中文摘要---------------------------------------------------------------------------4
3.英文摘要---------------------------------------------------------------------------6
4.研究背景---------------------------------------------------------------------------8
5.研究目的--------------------------------------------------------------------------17
6.實驗材料--------------------------------------------------------------------------18
7.實驗方法--------------------------------------------------------------------------22
8.實驗結果--------------------------------------------------------------------------35
9.討論--------------------------------------------------------------------------------43
10.結論 -----------------------------------------------------------------------------51
11.結果附圖 -----------------------------------------------------------------------52
12.參考文獻 -----------------------------------------------------------------------68

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