【作者】 张运阁；

【导师】 陶陆阳；

【作者基本信息】 苏州大学 ， 法医学， 2009， 硕士

【摘要】 目的:研究自噬/溶酶体途径对脑外伤后神经细胞死亡和神经功能障碍的影响并探讨其相关机制。方法:建立小鼠定量脑外伤(traumatic brain injury, TBI)动物模型,运用影响自噬体成熟的工具药3-甲基腺嘌呤(3-methyladenine,3-MA)、布雷菲德菌素(Brefeldin A,BFA)侧脑室(lateral cerebral ventricle)注射给药,对3-MA组、BFA组及生理盐水对照组用PI标记和体视学方法观测损伤区及其周边区(皮层与海马)神经细胞死亡情况,通过连续切片苏木精染色和体视学软件测量脑缺损体积(lesion volume, LV),并通过行为学试验方法(Motor test和Morris water maze)检测TBI引起的神经功能障碍,研究自噬对TBI引起的神经细胞死亡和神经功能障碍的影响;同时应用免疫组织化学和免疫印记法检测损伤侧的脑皮质与海马中自噬/溶酶体途径及凋亡信号通路相关蛋白Cathepsin-B,LC3,Beclin-1,Caspase-3,Bcl-2,Bax的表达情况,研究自噬/溶酶体途径影响TBI引起的神经细胞死亡和神经功能障碍的可能机制。结果:(1)PI阳性细胞计数:TBI后1 h和6 h,3-MA组与生理盐水组PI阳性细胞数均开始逐渐增加,12 h后明显增加,但两组之间无统计学差异,24 h阳性细胞数达高峰,且两组的PI阳性细胞数有统计学差异(P<0.05),TBI后48 h组仍有大量的PI阳性细胞,但3-MA组与生理盐水组比较无统计学差异;同样,BFA组与生理盐水组比较,TBI后24 h,两组的PI阳性细胞数有统计学差异(P<0.05)。(2)LV检测:与生理盐水组比较,3-MA组、BFA组脑外伤后LV显著减小(P<0.05);(3)行为学检测:与生理盐水组比较,3-MA组和BFA组在TBI后24 h均能改善运动功能(P<0.05),48 h以后差异不显著;3-MA组在TBI后7 d到10 d能改善学习记忆能力(P<0.05);(4)蛋白表达检测:TBI后24 h和48 h,3-MA组相对与生理盐水组Cathepsin-B, Caspase-3表达减少(P<0.05),Beclin-1/ Bcl-2的比值减小(P<0.05),Bcl-2 /Bax的比值增大(P<0.05);与生理盐水对照组比较,TBI后24 h和48 h BFA组LC3Ⅱ/LC3Ⅰ的比值减小(P<0.05)。结论:1.自噬/溶酶体途径参与了TBI后的病理生理过程。阻滞自噬体形成可以减少TBI引起的神经细胞死亡和脑组织缺损体积,并改善运动和学习记忆功能。2.自噬/溶酶体途径通过影响细胞凋亡信号通路来调节TBI引起的神经细胞死亡和神经功能障碍。更多还原

【Abstract】 Objective: The present study was sought to study the contribution of autophagy/lysosomal pathway to neuronal cell death and and neuronal dysfunctions after traumatic brain injury, and to explore its relevant mechanisms.Methods: Traumatic brain injury (TBI) model in mice was established quantitatively. Mice were pretreated with lateral cerebral ventricle infusion of two autophagic inhibitor 3-Methyladenine (3-MA) and Brefeldin A (BFA). TBI-induced cortical and hippocampal neuronal cell death was evaluated by intraperitoneal injection of propidium iodide. Lesion volume was estimated by campeachy dyeing and stereology microscopic observation, and a subset of mice were studied behaviorally (Motor test and Morris water maze) in our study to detect motor and cognitive function of the injured animals post-TBI. By Immunofluorescence, stereology microscopic observation and western blot analysis, changes of protein expression in autophagy/lysosomal pathway and apoptosis signal pathway were detected: Cathepsin-B, LC3, Beclin-1, Caspase-3, Bcl-2, and Bax in injuried cortex and hippocampus after TBI, and try to study the possible contribution of autophagy/lysosomal pathway to neuronal cell death and and neuronal dysfunctions after TBI.Results: (1) PI positive cell counts: PI-positive cells were found starting from 1 h and 6 h after TBI and increased rapidly at 12 h, peaked at 24 h, but decreased from 48 h. The number of PI positive cells in 3-MA or BFA group was signifcantly different from that in saline group at 24 h respectively (P<0.05). (2) Lesion Volume Measurements: the lesion volume was obviously reduced in 3-MA or BFA treated groups compared with the saline group (P<0.05). (3) Praxiology Experiment: 3-MA and BFA could significantly attenuated motor dysfunctions respectively in 24 h post-TBI (P<0.05), but it was not obviously different from that at later time stages; 3-MA could significantly attenuated learning and memory dysfunctions from 7 d to 10 d post-surgery campared with that in saline group (P<0.05). (4) Protein Expression: in 3-MA group, the mean ratios of the band intensities of cathepsin-B, caspase-3, beclin-1/bcl-2 significantly decreased (P<0.05) and a significant recovery of Bcl-2 /Bax (P<0.05) was observed at 24 h and 48h. Pretreatment with BFA resulted in a significant decrease of LC3-II/LC3-I levels from 24 h to 48 h (P<0.05).Conclusions:1. Autophagy/lysosomal pathway involved in the physiopathologic course of post-TBI. Blockage of autophagy/lysosomal pathway attenuated TBI-induced Neuronal cell death, decreased lesion volume, and improved motor and cognitive functions.2. Autophagy/lysosomal pathway regulated the TBI-induced neuronal cell death and neuronal dysfunction by influencing apoptosis signaling pathway.更多还原