創傷弧菌是一種伺機性的致病菌，感染創傷弧菌會造成致命性的敗血症及傷口感染，因感染後發病病程相當快速約入院後2-3天死亡，死亡率高達50%。創傷弧菌造成嚴重的病症主要是因為巨噬細胞受致病菌的毒殺，使人體無法保持抵抗力而遭受感染。先前的研究發現，創傷弧菌H毒素對巨噬細胞具有毒殺能力，且會使巨噬細胞走向細胞壞死。因此，本論文的目標為分析創傷弧菌H毒素引發巨噬細胞死亡的死亡途徑。由先前的研究已知鈣離子會引起細胞死亡訊號導致細胞死亡。本研究利用螢光染劑分析受創傷弧菌感染的巨噬細胞內的鈣離子，由結果發現巨噬細胞受創傷弧菌感染時，巨噬細胞外的鈣離子會大量進入到細胞內，當巨噬細胞內的鈣離子增加時會引發細胞走向程序性死亡。且巨噬細胞內鈣離子濃度增加會導致細胞內產生ROS，造成細胞的死亡。以抑制劑分析結果得知，藉由抑制巨噬細胞的NOX及CypD的功能，避免創傷弧菌H毒素所引發的死亡。同時，使用螢光染劑分析的結果顯示，巨噬細胞受創傷弧菌感染時，巨噬細胞會產生ROS及造成粒線體的損傷。由以上結果顯示，創傷弧菌H毒素會使鈣離子進入巨噬細胞內，進而產生ROS，造成粒線體損傷，引發巨噬細胞死亡。 Vibrio vulnificus is an opportunistic pathogen, and the bacteria can cause fatal septicemia and wound infections. V. vulnificus infections can lead to death in 2-3 days after hospitalization, with mortality rates as high as 50%. Our previous studies found that V.vulnificus H toxin has the ability to cause cell necrosis in macrophages. Therefore, the goal of this thesis is the analysis of pathway of V.vulnificus H toxin-induced macrophage death.Our previous studies showed that calcium ions cause cell death in V.vulnificus-treated macrophages. In this study, we showed that the V.vulnificus H toxin cause extracelluar calcium influx into cytoplasm of macrophages. By using inhibition analysis, inhibition of NADPH oxidase leaded to a reduction in V.vulnificus H toxin-induced cytotoxicity in macrophages. The H toxin also caused ROS generation and mitochondria damage in V.vulnificus-infected macrophages. These findings suggest that H toxin plays an important role in V.vulnificus infection.
