【作者】 王焕萍；

【导师】 严杰；

【作者基本信息】 浙江大学 ， 病原生物学， 2005， 硕士

【摘要】 背景和目的 钩端螺旋体（简称钩体）病是全世界流行的人兽共患病,致病性钩体侵入人体后能引起急性发热和全身各系统的疾病。钩体病的携带者主要是野生动物或家畜,尤其是啮齿动物、有袋动物、牛、猪和狗。其临床综合症包括亚临床感染、伴或不伴有脑膜炎的无黄疸的自限性发热、潜在性致命的以出血、黄疸、肾衰竭为表现的Weil’s综合症。虽然钩体病的致病因素如:脂多糖、脂蛋白、肽聚糖、热休克蛋白及鞭毛蛋白等可能与致病性有关,但确切的致病机制至今未明。 近年来报道,许多致病菌黏附宿主细胞后能引起Ca²⁺升高,Ca²⁺可以触发信号传导机制,诱导宿主细胞骨架重排,最终导致细菌内化入细胞。钩体内化细胞时能否引起Ca²⁺升高及感染细胞的结局如何,尚需要研究。 在以往工作基础上,本研究用Annexin V-FITC/PI染色和流式细胞术检测了活的或紫外杀死的56601株钩体对细胞凋亡的作用;采用Fluo-3/AM胞内游离Ca²⁺特异荧光标记激光共聚焦技术检测了不同毒力的钩体对宿主细胞胞内游离Ca²⁺水平的影响及Ca²⁺的来源:以[³H]1,3二磷酸磷脂酰肌醇（PIP₂）（[³H）PIP₂)为底物,检测了不同毒力的钩体磷脂酶C（PLC）的活性。 方法将新鲜培养或经紫外线直接照射60 min杀死的56601株钩体分别与Vero和J774A.1单层细胞共孵育1h,用Annexin V-FITC和propidium iodide（PI）染色,按AnnexinV试剂盒操作说明书,用流式细胞仪检测细胞凋亡情况。活细胞不被染料染色,早期凋更多还原

【Abstract】 Backgrand and Objective Leptospirosis is a spirochetal zoonosis that causes an acute febrile and systemic illness in humans caused by pathogenic Leptospira species. The leptospirosis carriers are mainly wild or domestic animals, especially rodents, small marsupials, cattle, pigs, and dogs. The clinical syndromes include subclinical infection, self-limited anicteric febrile illness with or without meningitis, and severe and potentially fatal Weil’s syndrome that manifests hemorrhage, jaundice, and renal failure. Although Leptospira virulence factors such as hemolysins, lipopolysaccharide, glycolipoprotein, peptidoglycan, heat shock proteins, flagellin, and others may contribute to the pathogenesis, their pathogenetic mechanisms have not been clearly understood.It is reported that many kinds of bacteria can induce the elevations of Ca²⁺ after adherence to host cell, and Ca²⁺ can trigger rapid and precise signals and spatially assemble host molecules for internalization. Whether Leptospira can induce the elevations of Ca²⁺ after adhenrence to cells and the result of the infected cells, both of them need studying.In our studies, the apoptosis induced by live or UV-killed L.interrogans strain 56601 was detected by flow cytometry using Annexin V-fluorescein isothiocyanate （FITC） binding propidium iodide （PI） staining, and the changes and the sourses of Ca²⁺ were detected by Laser Scanning Confocal Microscope with Fluo-3/AM staining. We assayed PLC activityusing [³H]PIP2 as a substrate for PLC as previously described.Methods J774A. 1 and Vero cells were infected with live or UV-killed L. interrogans strain 56601 for 60 min, and stained with Annexin V-fluorescein isothiocyanate （FITC） and propidium iodide （PI） according to the manufacturer’s instructions. Non-apoptotic live cells （Annexin V-FITC negative/PI negative）, early apoptotic cells （Annexin V-FITC positive/PI negative）, and late apoptotic cells （Annexin V-FITC positive/PI positive） were detected by flow cytometry. Vero and J774A.1 cells were loaded with fluorescent calcium indicator Fluo-3/AM for 30 min. Changes of intracellular free Ca²⁺ levels in Vero and J774A.1 cells infected with L.interrogans strain 56601, L.interrogans strain 56608 and L. biflexa strain Patoc I were detected by Laser Scanning Confocal Microscope after resting levels in normal cells were recorded. The cells loaded with Fluo-3/AM were excited at 488 nm, and the fluorescence emission was monitored at 530 nm. To detect the sourse of Ca²⁺, J774A.1 cells were pretreated with extracellular Ca²⁺ chelator EGTA, intracellular Ca²⁺ chelator BAPTA/AM before infected with L. interrogans strain 56601. J774A.1 cells were pretreated with PLC inhibitor U73122 and detected the changes of intracellular free Ca²⁺ levels infected with Linterrogans strain 56601. Using [³H]PIP2 as the substrate, the PLC activities in the culture supernatant, and cytoplasma and cytomembrane of the three strains of Leptospira were measured by isotope assay.Results Apoptosis rates in the Vero cells induced by live or UV-killed Linterrogans strain 56601 were 84.5% and 78.2%, respectively, while in J774A.1 cells under the same treatment were 34.5% and 30.9%, respectively. The resting fluorescence intensity of intracellular free Ca²⁺ levels in normal Vero and J774A.1 cells were 102.3±8.2% and 105.9±7.3%, respectively. The changes of fluorescence intensity percentage in the two cells infected with Lbiflexa strain Patoc I were fluctuated in range of 102.3±8.2%¹02.2±8.3% during the scan process. Two elevations in fluorescence intensity were observed in Vero and J774A.1 cells infected with L.interrogans strain 56601. The changes of fluorescence intensity percentage of the first peaks in Vero and J774A.1 cells were 430.5±35.7% and 747.5±18.5%, respectively, while the second peaks were 380.6±317.4% and 804.6±22.4%, respectively. In contrast to two elevations in Ca²⁺ seen in cells infected with L.interrogans strain 56601, only one elevation was observed in Vero and J774A.1 cells infected with L.inter更多还原