辛伐他汀及氯喹调节大鼠脑创伤后突触重建的研究

发布时间：2018-03-19 00:19

本文选题：自噬　切入点：脑创伤　出处：《华北理工大学》2015年硕士论文　论文类型：学位论文

【摘要】：目的探索脑创伤后的自噬水平神经突触再生之间的关系。从本实验通过应用自噬激动剂辛伐他汀和自噬抑制剂氯喹对脑创伤的大鼠进行干预,检查药物对大鼠海马区的自噬相关蛋白LC3和Beclin-1的表达水平的影响,以PSD-95和synaptophysin指示突触密度的高低。研究脑创伤后神经功能回复的机制,为脑创伤后临床治疗提供新的治疗靶点。方法按随机原则将200只Sprague Dawley雄性大鼠分为4组:1 Sham组(n=50)2TBI组(n=50)3辛伐他汀组(n=50)4氯喹组(n=50)。每组按手术后时间1d,3d,5d及7d分成四个亚组。使用Marmarou法,使用高空坠物打击的方法,建立大鼠弥漫性脑损伤模型。并进行以下各项检查:1使用HE染色法显示海马区神经元的形态;2免疫荧光检测LC3与Neu N的表达定位情况;3蛋白质印记法检测自噬相关蛋白LC3,Beclin-1和突触蛋白PSD-95,synaptophysin的表达水平。4 50只SD雄性大鼠在创伤后3-9d行Morris水迷宫测试。用q检验比较两组间的数据,组间使用单因素方差分析,P0.05作为差异具有统计学意义的界限。结果1 HE染色结果。sham组:海马神经元无明显的病理学改变,神经元清晰,整齐。TBI组:神经元数目减少,排列稀疏,水肿,细胞周围间隙和血管周围间隙较sham组变宽。辛伐他汀组:神经元数量减少较TBI组轻,轻微水肿,细胞周围间隙和血管周围间隙变宽较TBI组轻。氯喹组:与TBI组相比没有明显区别。2大鼠的空间学习记忆功能检测。TBI组在伤后的各组行为学参数,逃避潜伏期变长,穿越平台次数,平台所在象限路程比及时间比均显著减少。辛伐他汀组逃避潜伏期较TBI组变短,穿越平台次数,平台所在象限路程比及时间比均显著增加。氯喹组逃避潜伏期长于TBI组,穿越平台次数,平台所在象限路程比及时间比均减少。3 LC3 II的Western blot检测结果。Sham组:只可以检测到轻微量的蛋白。TBI组:LC3 II的蛋白表达量与Sham组比较,伤后1d出现明显升高(P0.05),3d达到表达最高点,创伤后7d的表达依然高于Sham组(P0.05)。辛伐他汀组:LC3 II表达创伤后1d和3d的表达量明显高于TBI组,有统计学意义(P0.05)。氯喹组:LC3 II蛋白表达TBI组相比较,在伤后3d和5d表达较低,具有统计学意义(P0.05)。4 Beclin-1的Western blot检测结果。Sham组:只可以检测到轻微量的蛋白,在各时像点无差异。TBI组:Beclin-1的蛋白表达量与Sham组比较,伤后1d出现明显升高(P0.05),3d达到表达最高点,创伤后7d的表达依然高于Sham组(P0.05)。辛伐他汀组:Beclin-1表达随时间变化的趋势和TBI组类似,在创伤后1d,3d和5d的表达量明显高于TBI组,有统计学意义(P0.05)。氯喹组:Beclin-1蛋白表达与时间的对应关系和TBI组类似,与TBI组相比较,在伤后3d表达较低,具有统计学意义(P0.05)。5 PSD-95的Western blot检测结果。Sham组:PSD-95蛋白表十分达明显,在各时像点无差异。TBI组:PSD-95的蛋白表达量与Sham组比较明显降低,之后几个时间点,PSD-95的表达会有缓慢的上升,但在7d依然明显低于sham组(P0.05)。辛伐他汀组:PSD-95表达随时间变化的趋势和TBI组类似,与TBI组相比,5d和7d的差别有统计学意义(P0.05)。氯喹组:PSD-95蛋白表达与TBI组类似,更为缓慢,在伤后7d表达较低,具有统计学意义(P0.05)。6 synaptophysin的Western blot检测结果。Sham组:synaptophysin蛋白表达十分明显,各时像点无差异。TBI组:synaptophysin的蛋白表达量与Sham组比较明显降低,之后几个时间点,synaptophysin的表达会有缓慢的上升,但在7d依然明显低于sham组(P0.05)。辛伐他汀组:synaptophysin表达随时间变化的趋势和TBI组类似,与TBI组相比,5d和7d的差别有统计学意义(P0.05)。氯喹组:synaptophysin蛋白表达与时间的对应关系和TBI组类似,与TBI组相比较,在伤后5d和7d较低,具有统计学意义(P0.05)。7 LC3与Neu N的免疫荧光检测结果。红色荧光显示自噬标记蛋白LC3的表达,绿色荧光显示神经元标记物Neu N的表达。结论辛伐他汀对大鼠的弥漫性脑创伤有保护作用,可以显著地提高神经功能的回复,海马区的突触相关蛋白表达水平提高。其可能的机制是通过加强自噬的水平,上调了神经网络的可塑性,使更多的突触得以重建,神经网络的完整性得到了更好的重建,而大鼠的学习记忆功能也得到了改善。
[Abstract]:Objective to explore the relationship between the level of autophagy in synaptic regeneration after traumatic brain injury. The intervention from the experiment on traumatic brain injury by using autophagy agonist simvastatin and chloroquine in rats, to examine the effects of drugs on rat hippocampus autophagy related protein LC3 and Beclin-1 expression, with PSD-95 and synaptophysin indicating synapse density of the mechanism. Reply to neural function after traumatic brain injury, for the treatment of brain trauma provide new therapeutic targets. Methods according to the principle of random 200 Sprague male Dawley rats were divided into 4 groups: 1 in group Sham (n=50) 2TBI group (n=50) 3 (n=50 4) simvastatin group chloroquine group (n=50). Each group according to the postoperative time of 1D, 3D, 5D and 7d are divided into four sub groups. Using the Marmarou method, using the method of falling down, establish the model of diffuse brain injury in rats. And the following tests: 1 using HE staining method According to the morphology of hippocampal neurons; localization of immunofluorescence detection of LC3 and Neu 2 N; 3 protein blot analysis of autophagy related protein LC3, Beclin-1 and synaptic protein PSD-95, synaptophysin expression level of.4 50 SD male rats at 3-9d after trauma underwent Morris water maze test. Using Q test to compare between the two groups data analysis using single factor variance between groups, as the limits of P0.05. The difference was statistically significant. The 1 group.Sham: the results of HE staining in hippocampal neurons without obvious pathological changes of neurons in.TBI group: clear, neat and reduce the number of neurons, sparse, edema, and blood vessel cells around the gap gap compared with the sham group variable wide. Simvastatin group: reduce the number of neurons was less than that in TBI group, mild edema, clearance and vascular cells around the gap becomes wider than the TBI group of light. There is no obvious difference between chloroquine group:.2 rats compared with TBI group The spatial learning and memory function of each behavior detection of group.TBI after injury the parameters, the escape latency became longer, the number of crossing platform, platform quadrant distance and time were reduced significantly. In simvastatin group compared with TBI group, the escape latency shortened, the times of crossing the platform, the platform quadrant time and distance were significantly increased than chloroquine group escape. The incubation period is longer than that of group TBI, the number of crossing platform, platform quadrant distance and time ratio were reduced by Western blot.3 LC3 II.Sham test results: group.TBI protein can be detected only a trace of light: the expression of LC3 II protein compared with Sham group, 1D increased significantly after injury (P0.05), 3D expression the highest point, the expression of 7D after trauma is still higher than that of Sham group (P0.05). Simvastatin group: LC3 II expression after trauma and expression of 1D 3D was significantly higher than that in TBI group, there was statistical significance (P0.05). Chloroquine group: LC3 II protein The expression of TBI compared to the group, at 3D after injury and the expression of 5D was lower, with statistical significance (P0.05) Western blot.4 Beclin-1.Sham group test results: only can detect light trace protein at different time points as there was no difference between group.TBI: the expression quantity of Beclin-1 compared with Sham group, 1D after injury there were significantly increased (P0.05), 3D expression reached the highest point, the expression of 7D after trauma is still higher than that of Sham group (P0.05). Simvastatin group: Beclin-1 expression trends over time and is similar to that of group TBI at 1D after trauma, the expression of 3D and 5D was significantly higher than that in group TBI, with statistical significance (P0.05). Chloroquine group: time and corresponding relation and similar to the TBI group of Beclin-1 protein expression, compared with the TBI group, the expression of 3D after injury was lower, with statistical significance (P0.05) Western blot.5 PSD-95.Sham group test results: PSD-95 protein was significantly different in the table is like a point, there was no difference between group.TBI: PSD-95 The amount of Sham group decreased significantly compared with the expressed protein, after some time point, the expression of PSD-95 is a slow rise in 7d, but still significantly lower than sham group (P0.05). Simvastatin group: PSD-95 expression trends over time and similar to the TBI group, compared with the TBI group, there was statistical significance in 5D and 7d the difference (P0.05). Chloroquine group: the expression of PSD-95 protein is similar with the TBI group, more slowly, after injury 7d expression was lower, with statistical significance (P0.05) Western blot.6 synaptophysin group.Sham results: the expression of synaptophysin is very obvious, like the point of no difference in.TBI group decreased significantly compared with the amount of the expression of synaptophysin protein in Sham group, after some time point, the expression of synaptophysin is a slow rise in 7d, but still significantly lower than sham group (P0.05). Simvastatin group: synaptophysin expression trends over time and is similar to that of group TBI and TBI Compared with statistical significance of 5D and 7d (P0.05). The difference between chloroquine group: time and corresponding relationship between TBI group and similar synaptophysin protein expression, compared with TBI, 5D and 7d after injury was lower, with statistical significance (P0.05) immunofluorescence detection results of.7 LC3 and Neu N. The red fluorescence showed that the expression of autophagy marker protein LC3, green fluorescence showed that the expression of neuronal markers Neu N. Conclusion simvastatin in rats with diffuse brain injury has a protective effect, can significantly improve the neurological function recovery, the expression level of synapse related protein in hippocampus increased. The possible mechanism is through enhancing the level of autophagy that raised the plasticity of neural network, the more able to reconstruct the synaptic integrity, the neural network has better reconstruction, and learning and memory function of rats was improved.

【学位授予单位】：华北理工大学
【学位级别】：硕士
【学位授予年份】：2015
【分类号】：R651.15

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