贝氏柯克斯体与单核细胞相互作用的研究

发布时间：2018-04-13 08:25

本文选题：贝氏柯克斯体 + Q热　；参考：《中国人民解放军军事医学科学院》2006年博士论文

【摘要】：贝氏柯克斯体是一种专性细胞内寄生的革兰阴氏性小球杆菌，为Q热的病原体。单核—巨噬细胞是贝氏柯克斯体关键宿主细胞。本研究拟通过细胞生物学和蛋白质组学等方法研究贝氏柯克斯体与宿主细胞(单核细胞)的相互作用，进一步探讨贝氏柯克斯体的致病机理，为寻找抗贝氏柯克斯体感染新型药物靶标、设计新型疫苗提供理论依据。本研究对贝氏柯克斯体进入单核细胞和在单核细胞内生长的规律进行了研究。结果发现，，人单核细胞(THP-1)对活贝氏柯克斯体的吞噬效率则显著低于其对γ射线灭活贝氏柯克斯体的吞噬。单核细胞内的活贝氏柯克斯体从第4天开始，数量逐渐增加；而灭活贝氏柯克斯体与单核细胞作用1天后，细胞内贝氏柯克斯体的量迅速减少，第5天后细胞内贝氏柯克斯体几乎被完全清除。在此基础上，本研究又对吞噬毒力株贝氏柯克斯体和未吞噬的单核细胞进行了比较蛋白质组学分析。对吞噬及未吞噬贝氏柯克斯体4小时后的单核细胞总蛋白样品双向电泳结果使用软件分析，总共发现158个差异蛋白，通过质谱鉴定一共确定了97个差异蛋白，多数蛋白表达为上调。对差异蛋白的功能进行分析，发现它们分别参与细胞的能量代谢、应激反应、细胞骨架运动、蛋白质和核酸合成代谢等。这些蛋白在单核细胞内的作用多数有利于贝氏柯克斯体在单核细胞内的生长繁殖和感染的建立。进一步研究这些差异蛋白在贝氏柯克斯体感染单核细胞中所起的作用，将为抗Q热药物、疫苗的的研制提供新的思路。本研究尝试建立了在鸡胚中培养至对数生长期的贝氏柯克斯体全菌蛋白参考图谱。并使用不同感染时期的小鼠血清，对贝氏柯克斯体全菌蛋白双向电泳分离蛋白进行了免疫印迹分析，用贝氏柯克斯体感染5天的血清，仅检测到2个蛋白点，用感染10天的血清检测到8个蛋白点，感染20天的血清检测到18个蛋白点，感染30天的血清共计检测到94个蛋白点，但是仅13个免疫反应蛋白被质谱分析鉴定。通过免疫蛋白质组学分析免疫反应蛋白的动态变化，可能发现新的免疫反应蛋白，为贝氏柯克斯体感染的早期诊断以及疫苗的研究提供新的靶标。
[Abstract]:Coxella Bayersoni is a specific intracellular parasite of Gram-Bacillus vulgaris, which is the pathogen of Q fever.Mononuclear-macrophages are the key host cells of Coxella bassii.In this study, we studied the interaction between Coxella bassii and host cells (monocytes) by means of cell biology and proteomics, and further explored the pathogenicity of Coxella bassii.In order to find a new drug target against Coxella bassii infection and design a new vaccine to provide theoretical basis.In this study, we studied the regularity of the entry and growth of Coxella Baysoni into monocytes.The results showed that the phagocytic efficiency of human monocyte THP-1 was significantly lower than that of 纬 -ray inactivated Coxella flexneri.The number of living Coxella in monocytes increased gradually from the 4th day, and the amount of Coxella Baysoni decreased rapidly after one day of inactivation of Coxella beersoni with monocytes.After 5 days, the intracellular Coxella bassii was almost completely removed.On this basis, we compared the proteomic analysis of the phagocytic virulence strain of Coxella Baysoni and the unphagocytic monocytes.A total of 158 differentially expressed proteins were found by using software to analyze the results of two-dimensional electrophoresis of total monocyte protein samples after 4 hours of phagocytosis and non-phagocytosis. 97 differential proteins were identified by mass spectrometry.Most proteins were up-regulated.By analyzing the function of differential proteins, it was found that they were involved in energy metabolism, stress response, cytoskeleton movement, protein and nucleic acid biosynthesis, etc.The effect of these proteins in monocytes is beneficial to the growth, reproduction and infection of Coxella bassii in monocytes.Further study on the role of these differential proteins in the infection of monocytes with Coxella bassii will provide a new idea for the development of anti-Q fever drugs and vaccines.In this study, a reference map of the whole bacterial protein of Coxella Baysoni was established from chicken embryo to logarithmic growth stage.Using mouse serum of different infection period, the two dimensional electrophoresis of the isolated proteins of Coxella beersoni was analyzed by Western blotting. Only two protein spots were detected in the sera infected with Coxella bassii for 5 days.Eight protein spots were detected in the serum of 10 days of infection, 18 spots in the serum of 20 days of infection, 94 spots in the serum of 30 days of infection, but only 13 immunoreactive proteins were identified by mass spectrometry.By analyzing the dynamic changes of immunoreactive proteins, new immunoreactive proteins may be found, which may provide a new target for the early diagnosis of Coxella bassii infection and the study of vaccines.
【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R378

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