肺炎链球菌溶血素致感染性脑损伤动物模型的建立

发布时间：2018-05-10 11:16

本文选题：肺炎链球菌溶血素 + 感染性脑损伤　；参考：《郑州大学》2011年硕士论文

【摘要】：背景和目的感染性脑损伤包括颅内感染(结核性脑膜炎、病毒性脑炎及化脓性脑膜炎)和颅外感染(中毒性痢疾及败血症、中毒性肺炎等)等所致的脑损伤,是儿科常见的危重症之一。细菌性感染是其主要的病因,以革兰阳性球菌感染最为常见,其中肺炎链球菌(Streptococcus pneumoniae, Spn)是婴幼儿中枢神经系统感染最常见的病原体之一,其广泛分布于自然界,常寄居于正常人的鼻咽腔中,儿童鼻咽部带菌率可高达24%-32%,研究显示Spn是细菌性肺炎及脑膜炎的主要病原体,同时也是儿童脑膜炎致死、致残率最高的病原体。Spn感染后患儿多表现为突然发病,出现惊厥、高热与昏迷等伴随症状,尸检可以发现脑组织肿胀、淤血,脑重量与容积均增加。报道称革兰氏阳性菌崩解后产生的溶血素(Pneumolysin, PLY)在感染性脑损伤中具有重要的作用,是最为重要的肺炎球菌细胞毒素,是肺炎球菌中唯一能破坏机械性宿主防御、补体和免疫应答的毒力因子。可引起广泛的毒性效应,如溶血、纤毛颤动、磷脂酶活化。PLY是一种属于巯基激活毒素家族的,所有肺炎链球菌都有的一种胞内蛋白。但目前PLY在感染性脑损伤中发生发展的确切机制尚未完全阐明。研究显示将含有肺炎链球菌菌落的生理盐水注入小鼠动脉内制成动物脑膜■炎模型后,血脑屏障(Blood-brain barrier, BBB)的紧密连接被破坏；另有研究发现,纯化的PLY在体外实验中可以诱导小胶质细胞和神经细胞凋亡。但是目前,国内外学者多采用腹腔、脑内直接注射肺炎链球菌或体外神经细胞培养等方法来研究肺炎链球菌及其溶血素致脑损伤的机制,直接用PLY注入实验动物体内的方法建立感染性脑损伤模型尚未见报道。本研究将根据人体疾病发生过程,客观模拟婴幼儿经血流途径致脑损伤感染过程建立动物模型,将革兰氏阳性菌的主要毒力因子—肺炎链球菌溶血素(PLY)直接注入实验动物动脉血流,使其到达脑组织,而引起病理改变,为更深入、全面和客观地研究PLY至感染性脑损伤的发病机制而打下基础。材料和方法选取健康普通级1月龄SD大鼠160只,体重100～120g,平均(105±10g),雌雄不限,随机平均分为PLY组和NS组,其中PLY组(实验组)：SD大鼠80只,颈内动脉注射PLY(剂量：0.2m1(7μg)),NS组(对照组)：SD大鼠80只,颈内动脉注射等体积生理盐水。根据不同观察时间点(注射后6h、12h、24h及48h),将PLY组SD大鼠分为4个亚组,每个亚组中分别有10只注射EB,测定不同时间点脑组织EB含量；注射EB组经右颈外静脉近心端注入2%EB(2m1/kg),推注时间大约1-2分钟。各组处理后,待观察至相应时间点,将大鼠麻醉行心脏灌注后断头取脑,用于制作脑组织标本,以备观察。脑组织EB含量测定采用甲酰胺法,不同时间点BWC测定采用干湿重法,此外,利用免疫组化法检测脑组织NSE、GFAP蛋白的表达。应用SPSS 16.0软件进行统计分析,采用的统计方法为t检验；检验水准为P0.05。结果 1.形态学观察：与NS组相比,采用HE染色光镜下观察,PLY组脑组织可见血管扩张、血管周围间隙的增宽、炎细胞浸润、神经元空泡变性、星形胶质细胞体积的增大肿胀、细胞核固缩等改变。 2.脑组织含水量和EB含量：注射6h后,PLY组脑组织含水量和EB含量开始增加,24h达到高峰,各时间点均显著高于NS组(p0.05)。 3.脑组织NSE和GFAP蛋白含量：注射6h后,PLY组NSE、GFAP蛋白开始增加,24h达高峰,48h仍维持较高水平,各时间点均显著高于NS组(p0.05)。 4.相关性分析：PLY组脑组织含水量和EB含量、NSE蛋白表达量和脑组织含水量、NSE蛋白表达量和EB含量、GFAP蛋白表达量和脑组织含水量、GFAP蛋白表达量和EB含量、NSE蛋白表达量和GFAP蛋白表达量均成正相关。结论 1.经大鼠颈内动脉注射PLY后,脑组织含水量增加,脑组织EB含量增加,血脑屏障(BBB)通透性增加,脑组织NSE蛋白、GFAP蛋白表达均增加,形态学方面发现细胞毒性和血管源性脑水肿病理改变,意味着血脑屏障破坏、脑水肿形成、神经元变性坏死及反应性星形细胞胶质化,表明该方法可以成功建立PLY致幼鼠感染性脑损伤模型。 2.在PLY致感染性脑损伤模型中,脑组织含水量和EB含量呈正相关,NSE蛋白表达量及GFAP蛋白表达量与脑组织含水量和EB含量呈正相关,提示它们均参与了BBB的破坏,而且可能促进了PLY所致大鼠感染性脑损伤的发生。
[Abstract]:Background and purpose
Infectious brain injuries include intracranial infection (tuberculous meningitis, viral encephalitis and suppurative meningitis) and brain injury caused by extracranial infection (toxic dysentery and sepsis, toxic pneumonia, etc.). It is one of the most common critical diseases in pediatrics. Bacterial infection is the main cause of the infection, and the most common infection of gram-positive cocci is the lung. Streptococcus pneumoniae (Spn) is one of the most common pathogens in the central nervous system infection in infants and children. It is widely distributed in nature and often resides in the nasopharynx cavity of normal people. The incidence of bacteria in the nasopharynx of children can be as high as 24%-32%. The study shows that Spn is the main pathogen of bacterial pneumonia and meningitis, and it is also the brain of children. Most of the children with the highest disability rate,.Spn infection, were sudden onset, convulsion, high fever, coma and other symptoms. Autopsy could find swelling of the brain tissue, blood stasis, and increased brain weight and volume.
It is reported that Pneumolysin (PLY), which is produced by the disintegration of Gram-positive bacteria, plays an important role in infectious brain damage. It is the most important pneumococcal cytotoxin. It is the only toxic factor in the pneumococcus that can destroy mechanical host defense, complement and immune response. It can cause extensive toxic effects, such as hemolysis and fibrinolysis. Hairy fibrillation, phospholipase activated.PLY is a kind of sulfhydryl activated toxin family, and all Streptococcus pneumoniae has a kind of intracellular protein. But the exact mechanism of the development of PLY in infectious brain damage has not yet been fully elucidated.
The study showed that the close connection of the blood brain barrier (Blood-brain barrier, BBB) was destroyed after the injection of the physiological saline containing Streptococcus pneumoniae into the mouse artery to make the animal meningoencephalitis model, and the purified PLY could induce the apoptosis of small gelatin cells and nerve cells in the experiment in vitro. The mechanism of brain injury caused by Streptococcus pneumoniae and its hemolysin is studied by intraperitoneal, direct injection of Streptococcus pneumoniae or in vitro neurocell culture in the brain. It is not reported that the model of infective brain damage can be established directly by PLY injection into experimental animals. This research will be objectively simulated according to the process of human disease. In the process of infantile infantile brain injury, the animal model is established. The main virulence factor of the Gram-positive bacteria - Streptococcus pneumoniae (PLY) is injected directly into the arterial blood flow of the experimental animal to make it reach the brain tissue and cause the pathological changes, so as to study the pathogenesis of PLY to infectious brain injury in a more thorough and objective way. And lay the foundation.
Materials and methods
160 healthy normal grade 1 month old SD rats, weight 100 ~ 120g, average (105 + 10g), and male and female, were randomly divided into group PLY and NS group, of which 80:SD rats in group PLY (experimental group) were injected PLY (dose: 0.2m1 (7 mu)), NS group (control group) 80 rats and internal carotid artery were injected with equal volume of physiological saline. At the time point (6h, 12h, 24h and 48h after injection), the PLY group SD rats were divided into 4 subgroups, and 10 of each group were injected with EB to determine the EB content in the brain tissue at different time points, and the injection EB group was injected with 2%EB (2m1/kg) by the right external jugular vein of the right external jugular vein for about 1-2 minutes. After treatment, the rats were treated to the corresponding time point and anesthetized rats. The rats were anesthetized. The brain tissue was taken from the head after heart perfusion and used to prepare the brain tissue specimens for observation. The EB content of the brain tissue was measured by formamide method and the dry wet weight method was used at different time points BWC. In addition, the expression of NSE and GFAP protein in the brain tissue was detected by immunohistochemical method. The statistical analysis was carried out with SPSS 16 software, and the statistical method adopted was t test. The test level is P0.05.
Result
1. morphological observation: compared with group NS, using HE staining light microscopy, the brain tissue of group PLY showed vascular dilatation, broadening of the perivascular space, inflammatory cell infiltration, vacuolation of neurons, swelling of astrocyte volume and nuclear condensation.
2. the water content and EB content of brain tissue: after injection of 6h, the water content and EB content of PLY group began to increase, and 24h reached its peak, which was significantly higher than that of NS group at all time points (P0.05).
3. the content of NSE and GFAP protein in brain tissue: after 6h injection, NSE, GFAP protein in group PLY began to increase, 24h reached its peak, and 48h still maintained a high level. All time points were significantly higher than those of the NS group (P0.05).
4. correlation analysis: the content of water content and EB content of brain tissue in PLY group, the expression of NSE protein and the water content of brain tissue, the expression of NSE protein and the content of EB, the expression of GFAP protein and the water content of brain tissue, the expression of GFAP protein and the content of EB, the expression of NSE protein and the expression of GFAP protein are all positive.
conclusion
1. after injecting PLY into the internal carotid artery, the water content of brain tissue increased, the content of brain tissue EB increased, the permeability of blood brain barrier (BBB) increased, the expression of NSE protein and GFAP protein in the brain tissue increased. The morphological aspects found the cytotoxicity and the pathological changes of vascular derived brain edema, which meant the destruction of blood brain barrier, the formation of brain edema and the degeneration and necrosis of neurons. The reactive astrocyte gliosis showed that this method could successfully establish the model of infective brain injury induced by PLY in young rats.
2. in the PLY induced brain injury model, the water content of brain tissue is positively correlated with the content of EB. The expression of NSE protein and the expression of GFAP protein are positively related to the content of brain tissue and the content of EB, suggesting that both of them are involved in the destruction of BBB and may promote the occurrence of infectious brain injury induced by PLY in rats.

【学位授予单位】：郑州大学
【学位级别】：硕士
【学位授予年份】：2011
【分类号】：R-332

【共引文献】