本文選題：自噬　切入點：腦創(chuàng)傷　出處：《華北理工大學》2015年碩士論文　論文類型：學位論文

【摘要】：目的探索腦創(chuàng)傷后的自噬水平神經突觸再生之間的關系。從本實驗通過應用自噬激動劑辛伐他汀和自噬抑制劑氯喹對腦創(chuàng)傷的大鼠進行干預,檢查藥物對大鼠海馬區(qū)的自噬相關蛋白LC3和Beclin-1的表達水平的影響,以PSD-95和synaptophysin指示突觸密度的高低。研究腦創(chuàng)傷后神經功能回復的機制,為腦創(chuàng)傷后臨床治療提供新的治療靶點。方法按隨機原則將200只Sprague Dawley雄性大鼠分為4組:1 Sham組(n=50)2TBI組(n=50)3辛伐他汀組(n=50)4氯喹組(n=50)。每組按手術后時間1d,3d,5d及7d分成四個亞組。使用Marmarou法,使用高空墜物打擊的方法,建立大鼠彌漫性腦損傷模型。并進行以下各項檢查:1使用HE染色法顯示海馬區(qū)神經元的形態(tài);2免疫熒光檢測LC3與Neu N的表達定位情況;3蛋白質印記法檢測自噬相關蛋白LC3,Beclin-1和突觸蛋白PSD-95,synaptophysin的表達水平。4 50只SD雄性大鼠在創(chuàng)傷后3-9d行Morris水迷宮測試。用q檢驗比較兩組間的數據,組間使用單因素方差分析,P0.05作為差異具有統(tǒng)計學意義的界限。結果1 HE染色結果。sham組:海馬神經元無明顯的病理學改變,神經元清晰,整齊。TBI組:神經元數目減少,排列稀疏,水腫,細胞周圍間隙和血管周圍間隙較sham組變寬。辛伐他汀組:神經元數量減少較TBI組輕,輕微水腫,細胞周圍間隙和血管周圍間隙變寬較TBI組輕。氯喹組:與TBI組相比沒有明顯區(qū)別。2大鼠的空間學習記憶功能檢測。TBI組在傷后的各組行為學參數,逃避潛伏期變長,穿越平臺次數,平臺所在象限路程比及時間比均顯著減少。辛伐他汀組逃避潛伏期較TBI組變短,穿越平臺次數,平臺所在象限路程比及時間比均顯著增加。氯喹組逃避潛伏期長于TBI組,穿越平臺次數,平臺所在象限路程比及時間比均減少。3 LC3 II的Western blot檢測結果。Sham組:只可以檢測到輕微量的蛋白。TBI組:LC3 II的蛋白表達量與Sham組比較,傷后1d出現(xiàn)明顯升高(P0.05),3d達到表達最高點,創(chuàng)傷后7d的表達依然高于Sham組(P0.05)。辛伐他汀組:LC3 II表達創(chuàng)傷后1d和3d的表達量明顯高于TBI組,有統(tǒng)計學意義(P0.05)。氯喹組:LC3 II蛋白表達TBI組相比較,在傷后3d和5d表達較低,具有統(tǒng)計學意義(P0.05)。4 Beclin-1的Western blot檢測結果。Sham組:只可以檢測到輕微量的蛋白,在各時像點無差異。TBI組:Beclin-1的蛋白表達量與Sham組比較,傷后1d出現(xiàn)明顯升高(P0.05),3d達到表達最高點,創(chuàng)傷后7d的表達依然高于Sham組(P0.05)。辛伐他汀組:Beclin-1表達隨時間變化的趨勢和TBI組類似,在創(chuàng)傷后1d,3d和5d的表達量明顯高于TBI組,有統(tǒng)計學意義(P0.05)。氯喹組:Beclin-1蛋白表達與時間的對應關系和TBI組類似,與TBI組相比較,在傷后3d表達較低,具有統(tǒng)計學意義(P0.05)。5 PSD-95的Western blot檢測結果。Sham組:PSD-95蛋白表十分達明顯,在各時像點無差異。TBI組:PSD-95的蛋白表達量與Sham組比較明顯降低,之后幾個時間點,PSD-95的表達會有緩慢的上升,但在7d依然明顯低于sham組(P0.05)。辛伐他汀組:PSD-95表達隨時間變化的趨勢和TBI組類似,與TBI組相比,5d和7d的差別有統(tǒng)計學意義(P0.05)。氯喹組:PSD-95蛋白表達與TBI組類似,更為緩慢,在傷后7d表達較低,具有統(tǒng)計學意義(P0.05)。6 synaptophysin的Western blot檢測結果。Sham組:synaptophysin蛋白表達十分明顯,各時像點無差異。TBI組:synaptophysin的蛋白表達量與Sham組比較明顯降低,之后幾個時間點,synaptophysin的表達會有緩慢的上升,但在7d依然明顯低于sham組(P0.05)。辛伐他汀組:synaptophysin表達隨時間變化的趨勢和TBI組類似,與TBI組相比,5d和7d的差別有統(tǒng)計學意義(P0.05)。氯喹組:synaptophysin蛋白表達與時間的對應關系和TBI組類似,與TBI組相比較,在傷后5d和7d較低,具有統(tǒng)計學意義(P0.05)。7 LC3與Neu N的免疫熒光檢測結果。紅色熒光顯示自噬標記蛋白LC3的表達,綠色熒光顯示神經元標記物Neu N的表達。結論辛伐他汀對大鼠的彌漫性腦創(chuàng)傷有保護作用,可以顯著地提高神經功能的回復,海馬區(qū)的突觸相關蛋白表達水平提高。其可能的機制是通過加強自噬的水平,上調了神經網絡的可塑性,使更多的突觸得以重建,神經網絡的完整性得到了更好的重建,而大鼠的學習記憶功能也得到了改善。
[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.

【學位授予單位】：華北理工大學
【學位級別】：碩士
【學位授予年份】：2015
【分類號】：R651.15

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