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

【摘要】：背景和目的 肺炎鏈球菌溶血素(pneumolysin, PLY)是肺炎鏈球菌(Streptococcus pneumoniae, S.pn)溶解釋放的一個主要的毒素,是S.pn的一個主要毒力因子,具有多種功能,能夠破壞血腦屏障的緊密連接、引起細胞溶解、刺激炎癥因子釋放、誘導(dǎo)細胞凋亡等,引起大腦嚴(yán)重損害。海馬及大腦皮層神經(jīng)元的凋亡使患兒學(xué)習(xí)、記憶能力下降,導(dǎo)致患兒智力低下,嚴(yán)重影響患兒生活質(zhì)量。近年來PLY致腦損傷機制備受關(guān)注,但多局限在體外研究,缺乏直接客觀的體內(nèi)研究。 腦損傷后既有細胞壞死又有細胞凋亡。凋亡誘導(dǎo)因子(apoptosis inducing factor, AIF)是在研究細胞凋亡時發(fā)現(xiàn)的,Susin等在1996年發(fā)現(xiàn)存在廣譜的胱天蛋白酶(caspase)抑制劑Z-VAD-FMK時,鼠肝細胞線粒體膜間J隙組分能夠使分離的HeLa細胞核染色體以非依賴于caspases的方式發(fā)生異常凝集和DNA大片段的形成,遂將其命名為AIF°AIF在感染性腦損傷中扮演者重要角色,但對其具體作用機制及調(diào)控機制的了解較少。有研究表明PLY可以通過線粒體釋放AIF途徑誘導(dǎo)腦細胞凋亡,但卻局限在體外研究。 本實驗通過頸內(nèi)動脈注射PLY制作感染性腦損傷動物模型,體內(nèi)研究細胞凋亡及AIF在S.pn所致腦損傷中的作用,為肺炎鏈球菌腦膜炎的臨床治療拓展新思路。 材料與方法 將80只幼鼠隨機分成溶血素組(PLY組,n=40),左頸內(nèi)動脈注射0.2mL(7μg)PLY；生理鹽水組(NS組,n=40),頸內(nèi)動脈注射等體積生理鹽水。兩組按注射后不同時間點分為6h、12h、24h、48h4個亞組,每亞組均10只。于預(yù)定時間點處死動物,制備腦組織標(biāo)本。干濕重法測腦含水量(brain watercontent, BWC),甲酰胺法測定伊文思蘭(Evan's blue, EB)含量,免疫組織化學(xué)技術(shù)(SP法)測定腦內(nèi)NSE、GFAP、AIF蛋白表達情況,原位末端標(biāo)記法(TUNEL法)檢測細胞凋亡。所有數(shù)據(jù)采用SPSS17.0軟件進行統(tǒng)計分析,顯著性水準(zhǔn)為a0.05。 結(jié)果 1形態(tài)學(xué)改變：PLY組左側(cè)腦體積較右側(cè)增大,腦組織腫脹發(fā)亮,腦膜緊張粘連,腦血管明顯充血。HE染色光鏡下觀察PLY組腦血.管間隙增寬,神經(jīng)元細胞、膠質(zhì)細胞不同程度腫脹、空泡變性,NS組沒有上述變化。 2 PLY組BWC、EB含量、NSE、GFAP(?)水平各時間點均明顯高于對照組。差異有統(tǒng)計學(xué)意義(p0.05)。 3 PLY組大腦皮層、海馬區(qū)可見細胞凋亡,隨時間逐漸增多,NS組可以見到少許凋亡細胞。PLY注射后6hAIF、凋亡細胞數(shù)均明顯增加,AIF在24h達高峰,凋亡細胞數(shù)在48h仍有較高表達,各時間點與對照組比較均有顯著性差異(p0.05)。 4 AIF含量與TUNEL檢測細胞凋亡結(jié)果呈正相關(guān),r=0.879,p0.05。 結(jié)論 1 PLY可以通過誘導(dǎo)神經(jīng)細胞凋亡方式導(dǎo)致腦損傷。 2 PLY通過線粒體釋放AIF途徑誘導(dǎo)神經(jīng)細胞凋亡。
[Abstract]:Background and purposeStreptococcus pneumoniae pneumolysin (plyyn) is a major toxin released by Streptococcus pneumoniae (S.pnnae) and a major virulence factor of S.pn. It has many functions, which can destroy the tight connection of blood-brain barrier and cause cell lysis.Stimulate the release of inflammatory factors, induce apoptosis, and cause serious brain damage.The apoptosis of hippocampal and cortical neurons decreased the learning and memory ability of the children, resulting in the mental retardation of the children, which seriously affected the quality of life of the children.In recent years, the mechanism of brain injury caused by PLY has attracted much attention, but most of them are confined to in vitro research and lack of direct and objective in vivo research.There are both necrosis and apoptosis after brain injury.Apoptosis-inducing inducing factor (AIFA) was found in the study of apoptosis of cells. The presence of Z-VAD-FMK, a broad-spectrum inhibitor of cystatin caspase, was found in 1996.The J gap between mitochondria of rat hepatocytes can cause abnormal agglutination of isolated HeLa nuclear chromosomes and the formation of large DNA fragments in a caspases independent manner, so it is named AIF 擄AIF which plays an important role in infectious brain injury.However, the specific mechanism of its action and regulation mechanism is less understood.Some studies have shown that PLY can induce apoptosis of brain cells through mitochondrial AIF pathway, but only in vitro.In this study, the animal model of infectious brain injury was made by injecting PLY into internal carotid artery. Apoptosis and the role of AIF in the brain injury induced by S.pn were studied in vivo, so as to develop a new idea for the clinical treatment of streptococcus pneumoniae meningitis.Materials and methods80 young rats were randomly divided into hemolysin group (n = 40), left internal carotid artery injection (0.2mL(7 渭 g) PLY, NS group (n = 40) and internal carotid artery injection (n = 40).According to different time points after injection, the two groups were divided into 4 subgroups at 6 h, 12 h, 24 h and 48 h, with 10 rats in each subgroup.Animals were killed and brain tissue specimens were prepared at a predetermined time point.Brain water content (BWCX), formamide (formamide method), Evanosaurus (EB1) content in brain were measured by dry and wet weight method. The expression of NSE-GFAPAIF protein in brain was detected by immunohistochemistry (SP method). Apoptosis was detected by in situ end labeling method (Tunel).All the data were analyzed by SPSS17.0 software, the significant level was a 0.05.Result1Morphologic changes: the volume of left brain was larger than that of right, brain tissue was swollen and bright, meningeal tense adhesion, cerebral vascular congestion and HE staining were observed under light microscope in PLY group.The interstitial space was widened, the neuronal cells and glial cells were swollen in varying degrees, and the vacuolar degeneration in NS group had no such changes.(2) the content of EB in PLY group was higher than that in the control group (P < 0.05).The level of each time point was significantly higher than that of the control group.The difference was statistically significant (P 0.05).3 apoptosis was observed in cerebral cortex and hippocampus in PLY group. A few apoptotic cells were observed in NS group at 6 h after injection. The number of apoptotic cells increased significantly, and the number of apoptotic cells reached its peak at 24 h, and the number of apoptotic cells remained high at 48 h.There was significant difference between each time point and the control group (P 0.05).4 there was a positive correlation between the content of AIF and the result of apoptosis detected by TUNEL.Conclusion1 PLY can induce brain injury by inducing neuronal apoptosis.2 PLY induced neuronal apoptosis through mitochondrial AIF release pathway.
【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】：R378

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1 潘斌;鄭少波;徐亞文;劉春曉;方平;;IgG在前列腺癌組織中的表達及臨床意義[J];實用醫(yī)學(xué)雜志;2012年08期

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