铜绿假单胞菌外膜囊泡的研究

发布时间：2018-07-18 12:30

【摘要】：第一部分PAO1外膜囊泡的提取的方法学建立及优化目的：建立并优化铜绿假单胞菌（Pseudomonas aeruginosa, PA）外膜囊泡（out membrane vesicle,OMV）体外的提取及纯化方法，为研究OMV的生物学活性及诱导感染发生的机制奠定基础。方法：以铜绿假单胞菌标准株PAO1为模式菌，分别采用分别采取超滤浓缩法和Optiripe密度梯度超速离心分离法提取PAO1的外膜囊泡，负染电镜检测形态及纯度；BCA法检测两种方法提取的OMV蛋白浓度并计算OMV产量。结果：超滤浓缩法提取的OMV含有较多杂质，产量为21.3ug/1010个细菌，密度梯度离心提取的OMV无明显杂质存在，产量为48.7ug/1010个细菌。结论：超滤浓缩法和Optiripe密度梯度超速离心法均可用于提取PA的外膜囊泡，但Optiripe密度梯度离心法提取的OMV的产率和纯度均高于超滤浓缩法提取OMV。第二部分PAO1外膜囊泡的成分及生物学活性检测目的:初步检测PAO1的OMV中所含蛋白，建立OMV体内体外感染模型，检测OMV的生物学活性方法:用SDS-PAGE电泳初步鉴定OMV中所含蛋白条带，建立OMV感染人支气管上皮细胞BEAS-2B，ELISA检测炎性因子IL-6、IL-8的表达水平；通过滴鼻方式建立OMV感染Bablc小鼠模型，提取小鼠肺组织，HE染色观察其炎性变化；通过多粘菌素B抵抗实验，检测OMV对细菌的保护作用。结果:SDS-PAGE电泳结果显示PAO1的OMV中含有多种蛋白条带，主要为10-100KDa大小的蛋白；PAO1的OMV能显著诱导Base-2B细胞表达炎性因子IL-6、IL-8，刺激Balbc小鼠肺组织发生明显的炎性变化，且能帮助PAO1抵抗多粘菌素B的杀害。结论:PAO1分泌的外膜囊泡为一个含有多种蛋白的混合体，具有诱导宿主细胞产生炎性反应以及保护感染细菌抵抗周围不利环境等多种生物学活性。第三部分lasB对PAO1外膜囊泡的影响目的:探讨lasB基因对PAO1的OMV的形态、分泌量、及防御功能的影响。方法:PCR鉴定PAO1标准株和lasB缺陷的PAO1株，用Optiripe密度梯度超速离心法提取PAO1标准株和lasB缺陷株的外膜囊泡，负染电镜观察两种外膜囊泡的形态、BCA法检测OMV蛋白浓度，计算OMV产率，，通过刚果红实验比较两种外膜囊泡弹性蛋白酶活性大小，建立OMV感染正常人外周血单个核细胞模型，通过计算细菌吞噬率检测OMV保护细菌抵抗单个核细胞吞噬的能力。结果:PAO1标准株和lasB缺陷株体外分泌的外膜囊泡形态一致，均为20-200nm的球形结构。与lasB缺陷株相比，PAO1标准株的OMV产率更高，所具有的弹性蛋白酶活性更强，且帮助细菌抵抗人外周血单个核细胞吞噬的能力更强。结论:lasB缺陷对PAO1体外分泌的外膜囊泡形态无影响，但lasB缺陷后，外膜囊泡的分泌量、所含弹性蛋白酶活性以及对细菌的保护能力均下降。
[Abstract]:Part I Methodology and Optimization of extracellular vesicles of PAO1 objective: to establish and optimize in vitro extraction and purification of Pseudomonas aeruginosa (PA) (out membrane vesicleus. It provides a basis for studying the biological activity of OMV and the mechanism of inducing infection. Methods: PAO1, a standard strain of Pseudomonas aeruginosa, was extracted by ultrafiltration concentration method and Optiripe density gradient ultracentrifugation method, respectively. The morphology and purity of PAO1 were detected by negative staining electron microscope. The concentration of OMV protein extracted by two methods was detected by BCA method and the production of OMV was calculated. Results: the concentration method of ultrafiltration method contained more impurities, the yield was 21.3ug/1010 bacteria, and the density gradient centrifugation method had no obvious impurity, and the yield was 48.7ug/1010 bacteria. Conclusion: both ultrafiltration concentration method and Optiripe density gradient ultracentrifugation method can be used to extract outer membrane vesicles of PA, but the yield and purity of OMV extracted by Optiripe density gradient centrifugation method are higher than that by ultrafiltration concentration method. The second part: detection of components and biological activity of PAO1 outer membrane vesicles objective: to detect the proteins contained in the OMV of PAO1 and to establish an in vivo and in vitro infection model of PAO1. Methods to detect the biological activity of OMV: SDS-PAGE electrophoresis was used to preliminarily identify the protein bands contained in OMV and to establish an Elisa to detect the expression of IL-6 and IL-8 in human bronchial epithelial cells infected with OMV. The Bablc mice model of OMV infection was established by nasal drip, and the inflammatory changes were observed by HE staining in lung tissue of mice, and the protective effect of OMV on bacteria was detected by polymyxin B resistance test. Results the results showed that there were many protein bands in the OMV of PAO1. OMV, which was mainly 10-100 KDa protein, could induce the expression of IL-6 and IL-8 in Base-2B cells, and stimulate the pulmonary tissue of Balbc mice. And can help PAO 1 to resist polymyxin B killing. Conclusion the adventitia vesicles secreted by 1% PAO1 are a mixture of many proteins and have many biological activities such as inducing inflammatory reaction of host cells and protecting the infected bacteria from the unfavorable environment around them. Part 3 effects of lasB on PAO1 adventitia vesicles objective: to investigate the effects of lasB gene on the morphology, secretion and defense function of PAO1. Methods PAO1 standard strain and PAO1 strain with lasB defect were identified by 1% PCR. The outer membrane vesicles of PAO1 standard strain and lasB defective strain were extracted by Optiripe density gradient ultracentrifugation method. The protein concentration of OMV was detected by negative staining electron microscope, and the yield of OMV was calculated. By comparing the activity of two kinds of outer membrane vesicle elastase with Congo red test, the peripheral blood mononuclear cells (PBMC) model of normal human infected with OMV was established, and the ability of OMV to protect bacteria against mononuclear cell phagocytosis was measured by calculating the phagocytosis rate of bacteria. Results the morphology of adventitia vesicles secreted by the standard strain of 1: PAO1 and the defective strain of lasB in vitro were identical and they were spherical structure of 20-200nm. Compared with the standard strain of lasB, the standard strain of PAO1 has higher yield of OMV, stronger activity of elastase and stronger ability to help bacteria resist phagocytosis of human peripheral blood mononuclear cells (PBMC). Conclusion the morphology of adventitia vesicles secreted by PAO1 was not affected by the defect of VlasB, but the secretion of adventitia, the activity of elastase and the ability of protecting bacteria were decreased after the defect of lasB.
【学位授予单位】：重庆医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R378.991

【相似文献】