【摘要】 目的　建立观察问号钩端螺旋体 (简称钩体 )黏附的双荧光染色法 ,探讨不同毒力钩体对细胞内游离Ca2 + 水平及细胞凋亡的影响。方法　以问号钩体黄疸出血群赖型 5 6 6 0 1株和双曲钩体三堡垄群patoc型PatocⅠ株抗血清为一抗、羊抗兔IgG荧光素F(ab) 2 和罗丹明F(ab) 2 片段为二抗的双荧光染色法 ,分别检测 5 6 6 0 1株和PatocⅠ株钩体对Vero、J774A .1细胞的黏附作用。采用fluo 3 AM胞内Ca2 + 特异荧光标记激光共聚焦技术 ,检测问号钩体 5 6 6 0 1株和波摩那群波摩那型 5 6 6 0 8株、双曲钩体PatocⅠ株作用的J774A .1细胞胞内游离Ca2 + 水平的变化。采用FITC annexinⅤ PI荧光标记流式细胞术 ,检测紫外线灭活前后的 5 6 6 0 1株钩体诱导Vero和J774A .1细胞凋亡的情况。结果　所建立的双荧光染色法能清晰地观察到强毒力的 5 6 6 0 1株钩体对Vero和J774A .1细胞的黏附 ,无毒力的PatocⅠ株钩体则否。正常J774A .1细胞胞内游离Ca2 + 基础值为 (1 0 5 .0± 7.0 ) %,PatocⅠ株钩体作用细胞的荧光强度变化百分数一直波动于 (1 0 2 .2± 5 .2 ) %。 5 6 6 0 1株钩体感染J774A .1细胞胞内游离Ca2 + 浓度迅速增高 ,呈现为双峰型曲线 ,其荧光强度变化百分数分别为 (74 7.5± 35 .7) %和 (80 4 .6±4 0 .8) %。 5 6 6更多还原

【Abstract】 Objective To establish a double fluorescence staining method for observing adhesion of L.interrogans, and to demonstrate intracellular free Ca 2+ level changes and apoptosis caused by different virulent leptospiral strains. Methods Changes of intracellular free Ca 2+ levels was examined by laser scanning confocal microscopy. Apoptosis of Vero and J774A.1 cells induced by L.interrogans strain 56601 was detected by flow cytometry with fluorescein labeling of FITC-Annexin V/PI. Results Intracellular free Ca 2+ levels in the J774A.1 cells infected with L.interrogans strain 56601 rapidly elevated with a double peak curve and the two peak fluorescence intensity change percentages were (747.5±35.7)% and (804.6±40.8)%, respectively. Intracellular free Ca 2+ levels in the J774A.1 cells infected with L.interrogans strain 56608 slowly risen with a single slope-like curve and its maximum of fluorescence intensity change percentages was (402.4±23.6)%, which was significantly lower than those in the cells infected with L.interrogans strain 56601 (P<0.01). Apoptosis rates in the Vero cells adhered with living and UV-killed L.interrogans strain 56601 were 84.5% and 78.2%, respectively, while in J774A.1 cells with the same treatment were 34.5% and 30.9% respectively. Conclusion The double fluorescence staining method established in this study can be used to observe adhesion of leptospira. There is a positive correlation between intracellular free Ca 2+ levels and virulence of the infected leptospiral strains. Apoptosis occurred immediately when virulent leptospiral strains touching the cells and the ligand molecule initiating apoptosis signal pathway probably existed in the surface of leptospira. [更多还原