小鼠颅内感染模型中agr系统对金黄色葡萄球菌毒力调控作用的探究

Ningxia Medical University Thesis for Application of Master’s Degree

The Accessory Gene Regulator (agr) Controls Staphylococcus Aureus Virulence in a Murine Intracranial Infection Model

Student’s Name:Li Dongzhi

Supervisor：Sun Tao

Subject Category:Medicine

Major:Surgery

Specialty: Basic and Clinical Study of Epilepsy

School: Ningxia Medical University

Completion Date:Apr 2014

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小鼠颅内感染模型中agr系统对金黄色葡萄球菌

毒力调控作用的研究

摘要

目的建立由金黄色葡萄球菌引起的小鼠颅内感染模型。研究小鼠颅内感染模型中agr系统对金黄色葡萄球菌毒力的调控作用。

方法选取6-8周龄雌性C57BL/6J小鼠作为本研究的实验对象，通过颅内定点注射金黄色葡萄球菌菌液的方法建立小鼠颅内感染模型，对照组小鼠注射等量的生理盐水。造模成功后，观察小鼠的症状，观察最早出现死亡的时间点及48h存活率。检测肛温、外周血白细胞计数及中性粒细胞百分比。取脑组织做病理切片，观察脑组织病理学改变。应用agr系统缺失的金葡菌菌株造模，观察不同菌株对小鼠致死性的差别。同时，在agr 系统缺失的金葡菌菌株中回复表达了α溶血素，并检测α溶血素在小鼠颅内感染模型的作用。全血杀伤实验检测金葡菌对全血的敏感性。

结果模型组小鼠出现反应迟钝、抽搐等症状，12h开始出现死亡，48h生存率为26.9%，中性粒细胞百分比明显增高，脑组织病理学显示感染处大量中性粒细胞浸润。对照组均未见上述症状和死亡，脑组织病理学检查正常。进一步的实验结果显示，由agr 系统缺失的金葡菌引起的颅内感染，小鼠死亡率明显降低。此外，还发现agr系统中调控分子RNAIII在颅内感染进程中至关重要。在agr系统缺失的金葡菌中回复表达了α溶血素之后，能部分恢复其致死性。

结论本文建立的小鼠金黄色葡萄球菌颅内感染模型特征稳定，可用于临床金葡菌颅内感染致病机制的研究。在小鼠颅内感染模型中，agr系统对金葡菌的毒力调控具有重要作用。

关键词：颅内感染，金黄色葡萄球菌，agr系统，RNAIII，α溶血素

The accessory gene regulator (agr) controls Staphylococcus aureus virulence in a murine intracranial infection model

ABSTRACT

Objective The purpose of this study is to construct the murine intracranial infection model induced by S.aureus and investigate the role of agr system in this model.

Methods S.aureus cells were injected with a micro-syringe into the mice frontal cortex to construct the animal model. The mice of control group were injected sterile saline instead of S.aureus. After injection, the vital sign and survival rate of mice were observed to evaluate the infection. The rectal temperature was monitored. At 4 hours and 8 hours after injection, hemogram was determined. Brain tissues were observed for pathology study at 12 hours. Histological examination was used to diagnose the pathological alterations of mouse tissues. The sensitivity of S.aureus to whole blood was evaluated by whole-blood killing assay.

Results The mice in the model group demonstrated clinical signs of illness including hunched posture, lethargy, spasm and finally dead. The earliest time point of death is 12h. Compared with saline group, the 48-hour mortality was significantly higher in model group which were injected S.aureus 8325-4. Meanwhile, neutrophil granulocyte infiltration in brain tissue has been observed in model group. Although the number of white blood cells and the rectal temperature of mice were not significantly altered in model group, the percentage of neutrophil was significantly increased at the different time points after injection of S.aureus.