肺炎链球菌溶血素致脑损伤细胞凋亡及凋亡诱导因子的表达及意义

发布时间：2018-04-06 01:22

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

【摘要】：背景和目的肺炎链球菌溶血素(pneumolysin, PLY)是肺炎链球菌(Streptococcus pneumoniae, S.pn)溶解释放的一个主要的毒素,是S.pn的一个主要毒力因子,具有多种功能,能够破坏血脑屏障的紧密连接、引起细胞溶解、刺激炎症因子释放、诱导细胞凋亡等,引起大脑严重损害。海马及大脑皮层神经元的凋亡使患儿学习、记忆能力下降,导致患儿智力低下,严重影响患儿生活质量。近年来PLY致脑损伤机制备受关注,但多局限在体外研究,缺乏直接客观的体内研究。脑损伤后既有细胞坏死又有细胞凋亡。凋亡诱导因子(apoptosis inducing factor, AIF)是在研究细胞凋亡时发现的,Susin等在1996年发现存在广谱的胱天蛋白酶(caspase)抑制剂Z-VAD-FMK时,鼠肝细胞线粒体膜间J隙组分能够使分离的HeLa细胞核染色体以非依赖于caspases的方式发生异常凝集和DNA大片段的形成,遂将其命名为AIF°AIF在感染性脑损伤中扮演者重要角色,但对其具体作用机制及调控机制的了解较少。有研究表明PLY可以通过线粒体释放AIF途径诱导脑细胞凋亡,但却局限在体外研究。本实验通过颈内动脉注射PLY制作感染性脑损伤动物模型,体内研究细胞凋亡及AIF在S.pn所致脑损伤中的作用,为肺炎链球菌脑膜炎的临床治疗拓展新思路。材料与方法将80只幼鼠随机分成溶血素组(PLY组,n=40),左颈内动脉注射0.2mL(7μg)PLY；生理盐水组(NS组,n=40),颈内动脉注射等体积生理盐水。两组按注射后不同时间点分为6h、12h、24h、48h4个亚组,每亚组均10只。于预定时间点处死动物,制备脑组织标本。干湿重法测脑含水量(brain watercontent, BWC),甲酰胺法测定伊文思兰(Evan's blue, EB)含量,免疫组织化学技术(SP法)测定脑内NSE、GFAP、AIF蛋白表达情况,原位末端标记法(TUNEL法)检测细胞凋亡。所有数据采用SPSS17.0软件进行统计分析,显著性水准为a0.05。结果 1形态学改变：PLY组左侧脑体积较右侧增大,脑组织肿胀发亮,脑膜紧张粘连,脑血管明显充血。HE染色光镜下观察PLY组脑血.管间隙增宽,神经元细胞、胶质细胞不同程度肿胀、空泡变性,NS组没有上述变化。 2 PLY组BWC、EB含量、NSE、GFAP(?)水平各时间点均明显高于对照组。差异有统计学意义(p0.05)。 3 PLY组大脑皮层、海马区可见细胞凋亡,随时间逐渐增多,NS组可以见到少许凋亡细胞。PLY注射后6hAIF、凋亡细胞数均明显增加,AIF在24h达高峰,凋亡细胞数在48h仍有较高表达,各时间点与对照组比较均有显著性差异(p0.05)。 4 AIF含量与TUNEL检测细胞凋亡结果呈正相关,r=0.879,p0.05。结论 1 PLY可以通过诱导神经细胞凋亡方式导致脑损伤。 2 PLY通过线粒体释放AIF途径诱导神经细胞凋亡。
[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 掳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.
【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】：R378

【引证文献】