本文選題：肺炎鏈球菌溶血素 + 感染性腦損傷��； 參考：《鄭州大學(xué)》2011年碩士論文

【摘要】：背景和目的 感染性腦損傷包括顱內(nèi)感染(結(jié)核性腦膜炎、病毒性腦炎及化膿性腦膜炎)和顱外感染(中毒性痢疾及敗血癥、中毒性肺炎等)等所致的腦損傷,是兒科常見的危重癥之一。細(xì)菌性感染是其主要的病因,以革蘭陽性球菌感染最為常見,其中肺炎鏈球菌(Streptococcus pneumoniae, Spn)是嬰幼兒中樞神經(jīng)系統(tǒng)感染最常見的病原體之一,其廣泛分布于自然界,常寄居于正常人的鼻咽腔中,兒童鼻咽部帶菌率可高達(dá)24%-32%,研究顯示Spn是細(xì)菌性肺炎及腦膜炎的主要病原體,同時(shí)也是兒童腦膜炎致死、致殘率最高的病原體。Spn感染后患兒多表現(xiàn)為突然發(fā)病,出現(xiàn)驚厥、高熱與昏迷等伴隨癥狀,尸檢可以發(fā)現(xiàn)腦組織腫脹、淤血,腦重量與容積均增加。 報(bào)道稱革蘭氏陽性菌崩解后產(chǎn)生的溶血素(Pneumolysin, PLY)在感染性腦損傷中具有重要的作用,是最為重要的肺炎球菌細(xì)胞毒素,是肺炎球菌中唯一能破壞機(jī)械性宿主防御、補(bǔ)體和免疫應(yīng)答的毒力因子�？梢饛V泛的毒性效應(yīng),如溶血、纖毛顫動、磷脂酶活化。PLY是一種屬于巰基激活毒素家族的,所有肺炎鏈球菌都有的一種胞內(nèi)蛋白。但目前PLY在感染性腦損傷中發(fā)生發(fā)展的確切機(jī)制尚未完全闡明。 研究顯示將含有肺炎鏈球菌菌落的生理鹽水注入小鼠動脈內(nèi)制成動物腦膜■炎模型后,血腦屏障(Blood-brain barrier, BBB)的緊密連接被破壞；另有研究發(fā)現(xiàn),純化的PLY在體外實(shí)驗(yàn)中可以誘導(dǎo)小膠質(zhì)細(xì)胞和神經(jīng)細(xì)胞凋亡。但是目前,國內(nèi)外學(xué)者多采用腹腔、腦內(nèi)直接注射肺炎鏈球菌或體外神經(jīng)細(xì)胞培養(yǎng)等方法來研究肺炎鏈球菌及其溶血素致腦損傷的機(jī)制,直接用PLY注入實(shí)驗(yàn)動物體內(nèi)的方法建立感染性腦損傷模型尚未見報(bào)道。本研究將根據(jù)人體疾病發(fā)生過程,客觀模擬嬰幼兒經(jīng)血流途徑致腦損傷感染過程建立動物模型,將革蘭氏陽性菌的主要毒力因子—肺炎鏈球菌溶血素(PLY)直接注入實(shí)驗(yàn)動物動脈血流,使其到達(dá)腦組織,而引起病理改變,為更深入、全面和客觀地研究PLY至感染性腦損傷的發(fā)病機(jī)制而打下基礎(chǔ)。 材料和方法 選取健康普通級1月齡SD大鼠160只,體重100～120g,平均(105±10g),雌雄不限,隨機(jī)平均分為PLY組和NS組,其中PLY組(實(shí)驗(yàn)組)：SD大鼠80只,頸內(nèi)動脈注射PLY(劑量：0.2m1(7μg)),NS組(對照組)：SD大鼠80只,頸內(nèi)動脈注射等體積生理鹽水。根據(jù)不同觀察時(shí)間點(diǎn)(注射后6h、12h、24h及48h),將PLY組SD大鼠分為4個(gè)亞組,每個(gè)亞組中分別有10只注射EB,測定不同時(shí)間點(diǎn)腦組織EB含量；注射EB組經(jīng)右頸外靜脈近心端注入2%EB(2m1/kg),推注時(shí)間大約1-2分鐘。各組處理后,待觀察至相應(yīng)時(shí)間點(diǎn),將大鼠麻醉行心臟灌注后斷頭取腦,用于制作腦組織標(biāo)本,以備觀察。腦組織EB含量測定采用甲酰胺法,不同時(shí)間點(diǎn)BWC測定采用干濕重法,此外,利用免疫組化法檢測腦組織NSE、GFAP蛋白的表達(dá)。應(yīng)用SPSS 16.0軟件進(jìn)行統(tǒng)計(jì)分析,采用的統(tǒng)計(jì)方法為t檢驗(yàn)；檢驗(yàn)水準(zhǔn)為P0.05。 結(jié)果 1.形態(tài)學(xué)觀察：與NS組相比,采用HE染色光鏡下觀察,PLY組腦組織可見血管擴(kuò)張、血管周圍間隙的增寬、炎細(xì)胞浸潤、神經(jīng)元空泡變性、星形膠質(zhì)細(xì)胞體積的增大腫脹、細(xì)胞核固縮等改變。 2.腦組織含水量和EB含量：注射6h后,PLY組腦組織含水量和EB含量開始增加,24h達(dá)到高峰,各時(shí)間點(diǎn)均顯著高于NS組(p0.05)。 3.腦組織NSE和GFAP蛋白含量：注射6h后,PLY組NSE、GFAP蛋白開始增加,24h達(dá)高峰,48h仍維持較高水平,各時(shí)間點(diǎn)均顯著高于NS組(p0.05)。 4.相關(guān)性分析：PLY組腦組織含水量和EB含量、NSE蛋白表達(dá)量和腦組織含水量、NSE蛋白表達(dá)量和EB含量、GFAP蛋白表達(dá)量和腦組織含水量、GFAP蛋白表達(dá)量和EB含量、NSE蛋白表達(dá)量和GFAP蛋白表達(dá)量均成正相關(guān)。 結(jié)論 1.經(jīng)大鼠頸內(nèi)動脈注射PLY后,腦組織含水量增加,腦組織EB含量增加,血腦屏障(BBB)通透性增加,腦組織NSE蛋白、GFAP蛋白表達(dá)均增加,形態(tài)學(xué)方面發(fā)現(xiàn)細(xì)胞毒性和血管源性腦水腫病理改變,意味著血腦屏障破壞、腦水腫形成、神經(jīng)元變性壞死及反應(yīng)性星形細(xì)胞膠質(zhì)化,表明該方法可以成功建立PLY致幼鼠感染性腦損傷模型。 2.在PLY致感染性腦損傷模型中,腦組織含水量和EB含量呈正相關(guān),NSE蛋白表達(dá)量及GFAP蛋白表達(dá)量與腦組織含水量和EB含量呈正相關(guān),提示它們均參與了BBB的破壞,而且可能促進(jìn)了PLY所致大鼠感染性腦損傷的發(fā)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.

【學(xué)位授予單位】：鄭州大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2011
【分類號】：R-332

【共引文獻(xiàn)】

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