【作者】 郑伟；

【导师】 严杰；

【作者基本信息】 浙江大学 ， 病原生物学， 2006， 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,我国也是钩体病流行的主要疫区。钩体通过皮肤黏膜,迅速进入血液,并在血液和组织内生长繁殖,造成机体损伤,引起一系列症状和体征。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。钩体在感染动物肾脏组织内长期生存并不断随尿排出,提示钩体必然具有侵入宿主细胞的能力,但确切的致病机制至今未明。 有文献报道,问号钩体可黏附并侵入细胞,进一步研究结果证实,强毒力和弱毒力问号钩体均能侵入J774A.1及Vero细胞,前者还可侵入细胞核,而无毒力的双曲钩体缺乏侵入细胞的能力。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触发信号传导机制,诱导宿主细胞骨架重排,尚需要研究。 在以往工作基础上,本研究用微丝（F-actin）的特异性标记物,罗丹明标记的鬼笔环肽（Phalloidin-TRITC）染F-actin,用抗微管蛋白α亚单位的小鼠一抗（Anti-Bovine α-Tubulin,Mouse Monoclonal）和荧光素标记的羊抗小鼠二抗（FITC-conjugated AffniPure Goat Anti-Mouse IgG）染微管（microtubule）,检测了不同毒力的钩体侵入宿主细胞时细胞骨架的改变情况,并用磷脂酶C（PLC）信号传导分子的特异性阻断剂U73122抑制磷脂酶C信号通路,观察其对细胞骨架改更多还原

【Abstract】 Background and Objective Leptospirosis is a pandemic spirochetalzoonosis, our country is also a epidemic area of Leptospirosis. Leptospire can rapidly invade the recirculating system through skin and mucosa, multiply in the blood or tissue, then cause the damage of organs induce sings and symptoms. 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. Leptospire can survive in the kidney of the infected animals and be discarded with urine, it means Leptospire has the ability to invade host cells, but the pathogenetic mechanisms have not been clearly understood.It is reported that Leptospira interrogans can adhere and invade host cells. Leptospira interrogans with higher virulence or lower virulence both can invade J774A.1 and Vero cells, the former even can invade the nucleus. Many kinds of bacteria can induce the elevations of Ca~2+ after adherence to host cell, and Ca~2+ can trigger signals transduction and induce the rearrangements of the cytoskeleton for internalization. Whether Leptospira can trigger the signals transduction and inducethe rearrangements of the cytoskeleton, both of them need studying.In our studies, the F-actin was visualized by fluorescence microscope with Phalloidin-TRITC staining;the microtubule was visualized by fluorescence microscope with mouse monoclonal anti-bovine a-Tubulin and FITC-conjugated afmipure goat anti-mouse IgG staining. We observed changes of the F-actin and the microtubule induced by Leptospira interrogans during invasion of the host cells. The inhibitor of PLC signal transduction pathway, U73122 was used to examine if it can block the rearrangements of the cytoskeleton of the host cells induced by Leptospira interrogans.Methods J774A.1 and Vero cells were cultivated into monolayer, theF-actin was stained with Phalloidin-TRITC for 60min and visualized by fluorescence microscope, cells were excited at 510nm, and the fluorescence emission was monitored at 620nm;the microtubule was stained with mouse monoclonal anti-bovine a-Tubulin and FITC-conjugated affhipure goat anti-mouse IgG for 60 min and visualized by fluorescence microscope, cells were excited at 490 nm, and the fluorescence emission was monitored at 520nm. Then cells were infected with LAnterrogans strain 56601 and LAnterrogans strain 56608, changes of the F-actin and the microtubule were observed by the same methods above. Cells were pretreated with PLC inhibitor U73122 and changes of the cytoskeleton were observed after the infection by Leptospira interrogans.Results After the Vero and J774A. 1 cells were infected with L. interrogansstrain 56601 and LAnterrogans strain 56608, there was no changes of the microtubule observed in both cell lines. The changes of F-actin were found in Vero cells but not in J774A.1 cells. The normal meshwork structure of the F-actin disappeared, they assembled like spots. The ratios of the rearrangements of the F-actin at 15min, 30min, 60min and 120min after the cells infected with LAnterrogans strain 56601 were different, they were 69.67 ± 2.02 %> 79.33+0.76%, 55.5±2.18%and 43.83+0.58 % respectively (P<0.0l), the results after the block of PLC signal transduction pathway were 24.67 +0.76 %> 27.83 + 4.54%, 21.5 + 1% and 19.83 ±4.01%respectively;the ratios of the rearrangements of the F-actin at 15min, 30min, 60min and 120min after the cells infected with LAnterrogans strain 56608 were also different, they were 62.17+0.76% ^73+2.5%.49.33 +1.61% and 35.33±2.02% (P<0.01), the results after the block of PLC signal transduction pathway were 21.67+1.61 % > 21.67+1.76%s 18.33+0.76% and 17.33+2.75%. The ratios of the rearrangements of F-actin at 15min and 30min were higher than those at 60min and 120min, and the meshwork structure of the F-actin reconverted somewhat after 60min postinfection. Ratio of the rearrangements of F-actin which triggered by LAnterrogans strain 56601 was a little higher than that of LAnterrogans strain 56608 (P<0.05), and at the same time postinfection ratio of the rearrangement of F-actin after the block of PLC was obviously lower than that before the block of PLC (P<0.01).Conclusion Rearrangements of the F-actin induced by LAnterrogansvaries with cells. There was a positive correlation between the ratio of the rearrangements of F-actin and virulence of the LAnterrogans strains. LAnterrogans invades the host cells mostly during 15min to 30min. The rearrangements of the F-actin can be partly inhibited by the PLC inhibitor U73122.更多还原