【作者】 张皓；

【导师】 严杰；

【作者基本信息】 浙江大学 ， 病原生物学， 2010， 硕士

【摘要】 目的:了解问号钩端螺旋体(简称钩体)对宿主细胞及细胞外基质(ECM)分子的黏附作用及其差异。了解多种宿主细胞ECM分子的种类及其分布差异。方法:建立问号钩体黄疸出血群赖型56601株对J774A.1、L929和Vero细胞感染模型,采用Fontana镀银染色法及显微镜检查法,了解问号钩体56601株黏附细胞ECM的效果。建立ELISAs,检测问号钩体56601株黏附ECM分子层粘连蛋白(LN)、纤维纤连蛋白(FN)、核心蛋白多糖(DEN)及胶原蛋白1、2、4(COL1,2,4)的作用,采用竞争性抑制试验对上述实验结果进行验证。采用免疫荧光染色法和激光共聚焦显微镜观察ECM分子FN、LN、DEN、COL1～4在THP-1、J774A.1、HEK293、NRK、COS-7、NRK-49F、L-02、IAR-20和HUVEC细胞中的分布。建立各种ELISAs定性验证以上结果并定量检测以上ECM分子在各细胞株的含量结果问号钩体56601株能以一端或两端黏附于L929、J774A.1和Vero细胞的ECM部位。问号钩体56601株可与上述6种ECM分子发生黏附,其中对COL1、LN、COL4的黏附作用较强。问号钩体56601株分别与上述6种ECM分子预孵育后,对相应ECM分子的黏附作用明显下降。各细胞ECM分子种类和含量存在明显差异。人单核-巨噬细胞THP-1和小鼠单核-巨噬样细胞J774A.1仅含有FN。人肾上皮细胞HEK293含有FN和COL4,大鼠肾上皮细胞NRK含有FN、LN,其中FN含量较高。猴肾成纤维细胞COS-7含有LN、COL3和COL4,其中LN和COL4含量较高。大鼠肾成纤维细胞NRK-49F含有FN、LN、DEN、COL3和COL4,其中FN、DEN和COL4含量较高。人肝上皮细胞L-02含有FN、LN、COL3和COL4,其中FN和COL4含量较高,而小鼠肝上皮细胞IAR-20仅含有COL4。人静脉内皮细胞HUVEC含有FN、LN、COL3和COL4。结论ECM是问号钩体黏附宿主细胞的主要部位。LN、FN、DEN、COL1、COL2、COL4均是问号钩体黏附的受体分子,但问号钩体对不同ECM分子的黏附效果有一定差异。不同的ECM分子分布在不同的问号钩体宿主细胞中,且含量存在明显差异。更多还原

【Abstract】 Objective: To determine the ability and diversity of Leptospira interrongans adhering to extracellular matrix (ECM) of host cells, and to determine the distribution and content of diferent extracellular matrix (ECM) molecules in various host cells of Leptospira interrongan.Methods Models of L.interrongans serogroup Icterohaemorrhagiae serovar lai strain 56601 infecting J774A.1, L929 and Vero cells were established, and Fontana silver impregnation method plus microscopy were applied to demonstrate the effect of L.interrongans strain 56601 adhere extracellular matrix (ECM) of the cells. ELISAs were established to detect the effects of L.interrongans strain 56601 adhere to ECM molecules laminin (LN), fibronectin (FN), decorin (DEN) and collagenl, 2, 4 (COL1, 2, 4). A competitive inhibition test was performed to verify the results of adhesion mentioned above. Immunity fluorescence dyeing method and confocal laser microscopy were used to observe the distribution of FN, LN, DEN and COL1～4 in THP-1, J774A.1, HEK293, NRK, COS-7, NRK-49F, L-02, IAR-20 and HUVEC cells. Different kinds of ELISAs were established to verify the results and detect the content of these ECM molecules in different cells.Results L.interrongans strain 56601 could use its one or two bodies to adhere the positions where were the ECMs of L929, J774A.1 and Vero cells. L.interrongans strain 56601 could adhere each the six ECM molecules in which the adhering effects to COL1, LN, COL4 were relatively stronger. The effects of L.interrongans strain 56601 adhering to the six ECM molecules were remarkably decreased after the microbe was pre-incubated with each the molecules. The distribution and content of different ECM molecules in host cells are various. THP-1 and J774A.1 cells contain FN only. HEK293 cells cotain FN and COL4. NRK contain FN and LN, the content of FN is high.. COS-7 contain LN, COL3 and COL4, the content of LN and COL4 is high. NRK-49F contain FN, LN, DEN, COO and COL4, the content of FN, DEN and COL4 is high. L-02 contain FN, LN, COL3 and COL4, the content of FN and COL4 is high. IAR-2 contain COL4, HUVEC contain FN, LN, COL3 and COL4, the content of FN and COL4 is high.Conclusion ECM is the position of L.interrongans adhering to host cells. LN, FN, DEN, COL1, COL2, COL 4 are the receptor molecules of adhesion secreted by L.interrongans, but the different ECM molecules show different adhered effects. The distribution of different ECM molecules in host cells of Leptospira interrongan is quite different, and the content is also distinguishing.更多还原