【作者】 白方方；

【导师】 张晓华；

【作者基本信息】 中国海洋大学 ， 微生物学， 2010， 博士

【摘要】 哈维氏弧菌(Vibrio harveyi)是一种革兰氏阴性、发光的海洋细菌。近年来,哈维氏弧菌已成为海水养殖动物(鱼、虾)的重要致病菌,其引起的弧菌病给海水养殖业造成了巨大的经济损失。目前,哈维氏弧菌的致病机理还不完全清楚。对哈维氏弧菌进行深入研究,对于我国水产养殖业的健康发展具有重要意义。本文首先研究了哈维氏弧菌对牙鲆各组织的侵染作用。用哈维氏弧菌VIB645感染牙鲆后,发病牙鲆的肾、脾、肝、鳃、脑和肠道等组织都发生明显的病理变化；用荧光免疫组化法对哈维氏弧菌在牙鲆中的分布进行了组织定位,发现牙鲆的肝、脾、肾和脑等组织中哈维氏弧菌呈阳性反应；通过扫描电镜技术研究了哈维氏弧菌对牙鲆红细胞的作用,结果表明哈维氏弧菌作用4h后牙鲆红细胞出现棘状突起,作用8h后出现起皱现象,作用12h时细胞裂解。哈维氏弧菌溶血素(Vibrio harveyi hemolysin,VHH)被认为是哈维氏弧菌对鱼类的重要致病因子,而活性位点突变的VHH溶血素(VHH突变蛋白)失去了溶血活力、磷脂酶活力和对鱼的致病性。本研究深入探讨了VHH溶血素对牙鲆红细胞和鳃细胞系FG-9307的作用。以VHH溶血素分别作用于牙鲆红细胞和鳃细胞,用台盼蓝染色法计算细胞存活率,发现牙鲆红细胞对VHH溶血素的敏感性远高于牙鲆鳃细胞。以VHH溶血素和VHH突变蛋白作用于牙鲆红细胞,电镜观察结果表明,经VHH溶血素作用后牙鲆红细胞表面首先发生棘皮化,随后细胞膜内陷,质膜破裂,最终细胞裂解,而VHH突变蛋白并不引起红细胞形态的明显变化。以VHH溶血素作用于牙鲆鳃细胞系,电镜观察结果表明牙鲆鳃细胞溶解严重,并形成凋亡小体；DAPI染色法进一步观察到受VHH溶血素侵染的牙鲆鳃细胞核体积变小,出现凋亡小体；细胞周期分析表明,VHH溶血素作用后的牙鲆鳃细胞的G]期比例及凋亡率增加；另外,VHH溶血素作用后牙鲆鳃细胞的原位末端缺口标记(TdT-mediated dUTP nick end labeling,TUNEL)的细胞阳性率增加,且半胱天冬氨酸酶-3(caspase-3)活力增加；这些结果表明,VHH溶血素通过caspases-3激活的细胞凋亡途径杀死牙鲆鳃细胞。通过免疫胶体金技术分析了VHH溶血素在牙鲆红细胞和鳃细胞中的分布,结果表明大量的VHH溶血素分布于细胞质膜及内膜系统上；而VHH突变蛋白在红细胞膜上也有少量分布。对腹腔注射VHH溶血素的牙鲆进行组织病理分析,结果表明受侵染的牙鲆肾、肝和肠道等有明显的组织病理变化,而VHH突变蛋白侵染的牙鲆无明显的组织病理变化；对VHH溶血素侵染的牙鲆各组织进行免疫组化分析显示,VHH溶血素主要分布在肠上皮细胞和鳃上皮细胞,说明肠道和鳃可能是哈维氏弧菌侵染牙鲆的重要部位。本研究深入探讨了哈维氏弧菌的致病机制,进一步阐明了TLH/VHH类溶血素的作用机理,对发展哈维氏弧菌的病害防治技术具有重要的指导意义。更多还原

【Abstract】 Vibrio harveyi is a Gram-negative, luminous marine bacterium, which is widely distributed in marine environment. This organism is a major pathogen of cultured penaeid shrimp, and has also been associated with disease in fish. The disease caused by V.harveyi has become a serious problem in marine aquaculture.However, we know little about the pathogenicity mechanism of V. harveyi.After the flounder were infected with V. harveyi VIB645, the histopathological changes of the diseased fish were studied, and it was demonstrated that the kidney, spleen, liver, gill, brain and intestine of the infected flounder had serious histopathological changes.The localization of V. harveyi on different tissues of flounder was also studied by indirect fluorescent antibody technique (IFAT) that used rabbit anti-V.harveyi serum, and we found that positive reaction with V. harveyi were identified in kidney, spleen, liver and brain of the infected flounder. Moreover, the ultrastructure changes of flounder erythrocytes were observed with scanning electron microscopy. The results demonstrated that erythrocyte membranes formed thin tubular protrusions after infected with V. harveyi for 4 h. When the incubation time attained to 8 h, the membrane corrugations were observed. The erythrocytes were finally lysed after 12 h incubation.Vibrio harveyi hemolysin (VHH) is considered a major pathogenic virulence factor to fish. However, the VHH activity-site mutant lost all the hemolytic and phospholipase activities as well as pathogenicity.In this study, the effect of VHH on erythrocytes and a gill cell line from flounder was elucidated. Erythrocyte membranes formed thin tubular protrusions immediately after exposure to VHH,and membrane corrugations were evident after extended incubation.In contrast, the mutant VHH did not induce any gross morphological changes.With VHH-treated FG-9307 cells,a cell line derived from flounder gill, destruction of organelles and formation of features resembling apoptotic bodies were observed. Apoptotic features, such as chromatin condensation and apoptotic bodies, were observed in VHH-treated FG-9307 cells using DAPI staining. Moreover, cell cycle analysis showed that VHH increased the proportion of cells in G1 phase. In addition, VHH significantly increased the percentage of apoptosis, the number of TUNEL positive apoptotic cells, and the caspase-3 activity in FG-9307 cells when compared with the untreated controls. These data suggested that VHH killed the cells through apoptosis via the caspase activation pathway. Immunogold staining showed that a large amount of VHH was deposited on the membranes and membrane debris of erythrocytes and FG-9307 cells after treatment with VHH. After flounder were intraperitoneally injected with VHH, the histopathological changes of the diseased fish were studied, and the results demonstrated that the kidney, liver and intestine of the infected flounder had serious histopathological changes. The results of immunohistochemistry illustrated that intense immunoreactive VHH was mainly localized to the intestinal and gill epithelial cells, indicating that intestine and gill may be the main target-sites for invasion by V. harveyi. This study facilitated the understanding of pathogenesis of V. harveyi and the mode of action of VHH/TLH-like hemolysin and would be helpful to the development of diseases control and prevention technique against V. harveyi.更多还原