【摘要】：目的：本研究擬通過觀察毛冬青甲素（IlexoninA，IA）對大鼠腦缺血再灌注后Wnt信號通路的調(diào)控作用和神經(jīng)再生的情況，探討IA促進神經(jīng)元再生及腦保護作用的可能機制。 方法：SD大鼠(雄性)隨機分為正常組、假手術(shù)組（手術(shù)過程同模型組，只插入線栓不阻塞大腦中動脈）、模型組、IA治療組，各組又分為再灌注1d、3d、7d、14d四個亞組，每組各9只。用線栓阻塞大腦中動脈制作大鼠局灶性腦缺血再灌注模型，通過Longa法對大鼠的神經(jīng)功能進行評分；通過腦組織TTC染色觀察腦缺血再灌注損傷后的腦梗死情況；通過免疫熒光雙標(biāo)的方法觀察缺血周邊組織Brdu/nestin、Brdu/NueN雙標(biāo)陽性細(xì)胞數(shù)；通過免疫熒光、western bolt、RT-PCR檢測經(jīng)典Wnt信號通路相關(guān)因子β-catenin、GSK3β、Axin、LEF-1的表達(dá)情況。 結(jié)果：（1）神經(jīng)功能缺損的癥狀于腦缺血再灌注后第1d明顯可見，癥狀隨時間的推移有所改善。神經(jīng)功能缺損的評分治療組較模型組低，治療組第3d和7d與模型組對應(yīng)的時間點相比差異有顯著性意義（P0.05）。（2）缺血周邊組織細(xì)胞情況：模型組和治療組Brdu/nestin、Brdu/NeuN雙標(biāo)陽性細(xì)胞于再灌注后第3d表達(dá)明顯增多，第7d開始下降，到第14d時仍有少量表達(dá)，3d表達(dá)最多；其中治療組Brdu/nestin、Brdu/NeuN雙標(biāo)陽性細(xì)胞于缺血再灌注后第3d、7d與模型組相應(yīng)時間點比較有顯著性差異（P0.05）（3）缺血周邊組織β-catenin的表達(dá)情況：模型組入核β-catenin陽性細(xì)胞數(shù)量再灌注后第1d開始表達(dá)，第3d表達(dá)達(dá)高峰，第7d開始下降，第14d仍有表達(dá)；治療組各時段入核β-catenin陽性細(xì)胞表達(dá)明顯多于模型組，其中第3d、7d與模型組相對應(yīng)的時間點比差異有統(tǒng)計學(xué)意義（P0.05）。（4）免疫蛋白印跡：①β-catenin蛋白表達(dá)情況：模型組β-catenin蛋白表達(dá)于缺血再灌注后1d開始增多，3d表達(dá)最多，其條帶密度明顯增高，而后逐漸下降，，至14d仍有表達(dá)，治療組β-catenin蛋白較模型組明顯增高，1d、3d、7d與模型組相應(yīng)時間點比較有顯著性差異（P0.05）。β-catenin蛋白表達(dá)情況所得結(jié)果與免疫熒光結(jié)果相吻合。②GSK3β蛋白表達(dá)情況：模型組GSK3β蛋白隨時間的延長，表達(dá)逐漸減弱，治療組GSK3β蛋白各時段表達(dá)明顯減少，條帶較淡，1d、3d、7d、14d與模型組相應(yīng)時間點比較有顯著性差異（P0.05）（5）RT-PCR法檢測：①Axin mRNA表達(dá)情況：模型組Axin mRNA隨時間的延長表達(dá)逐漸減弱，治療組Axin mRNA表達(dá)較模型組各時段有所減少，條帶較淡，其中3d、7d與模型組相應(yīng)時間點比較有顯著性差異（P0.05）②LEF1mRNA表達(dá)情況：模型組LEF1mRNA于缺血再灌注后1d表達(dá)增多，3d表達(dá)最多，而后逐漸下降，至14d仍有表達(dá)，治療組LEF1mRNA表達(dá)較模型組高，其中第3d、7d、14d與模型組相對應(yīng)時間點比差異有統(tǒng)計學(xué)意義（P0.05）. 結(jié)論：毛冬青甲素對大鼠腦缺血再灌注后的神經(jīng)功能有明顯改善作用，其機制可能是通過上調(diào)Wnt通路核心分子β-catenin的表達(dá)及促進β-catenin蛋白入核，下調(diào)抑制性因子GSK3β、Axin的表達(dá)，激活下游靶基因LEF1的轉(zhuǎn)錄，從而激活Wnt信號通路，促進神經(jīng)再生，保護受損腦組織。
[Abstract]:Objective: To study the effect of Ilex ininA (IA) on the control and regeneration of Wnt signaling pathway after cerebral ischemia-reperfusion in rats, and to explore the possible mechanism of IA to promote the regeneration of neurons and the protection of the brain. Methods: SD rats (male) were randomly divided into the normal group, the sham-operation group (the same model group as the same model group, only the plug was inserted into the middle cerebral artery of the brain), the model group and the IA treatment group, each group was divided into four sub-groups of reperfusion 1d, 3d, 7d and 14d, each group of 9 Methods: The focal cerebral ischemia-reperfusion model of the cerebral artery was blocked by a line bolt, the neurological function of the rat was scored by the Longa method, and the cerebral infarction after the cerebral ischemia-reperfusion injury was observed by the TTC staining of the brain tissue; and the Brdu/ nesti of the ischemic peripheral tissue was observed by the method of immunofluorescence double-label. N, the number of Brdu/ NguN double-labeled positive cells; by means of immunofluorescence, western blot, RT-PCR, the expression of the classical Wnt signaling pathway-related factors, such as P-catenin, GSK3, Axin, LEF-1, was detected. Results: (1) The symptoms of neurological deficit are clearly visible in the first day after cerebral ischemia and reperfusion, and the symptoms over time Compared with the model group, the score of the neurological deficit in the treatment group was lower than that of the model group, and the difference of the time points corresponding to the model group was significant (P0. (2) The expression of Brdu/ nestin, Brdu/ NeuN double-labeled positive cells in the model group and the treatment group was significantly increased in the third day after reperfusion. The expression of Bdu/ NeuN double-labeled positive cells in the third day, the 7th day and the corresponding time point of the model group was significantly different (P0.05). In the treatment group, the expression of catamenin positive cells was significantly higher than that of the model group, among which the time-point ratio of the third day and the 7th day to the model group was significantly higher than that of the model group (P0. 05). (4) immunoblotting: the expression of the antigen-cattenin protein in the model group began to increase at the beginning of 1 d after the ischemia-reperfusion, and the expression of 3-d was the most, the band density of the model group was significantly increased, and then gradually decreased to 14 d. Compared with the model group, there was a significant difference in the group of P-catenin protein in the treatment group, and the corresponding time point of 1d, 3d, and 7d was significantly different from that of the model group (P0. 05). Results of the expression of the antigen-cattenin protein and the result of immunofluorescence The expression of GSK3 protein in the model group was consistent with the time, the expression of GSK3 and the expression of GSK3 protein in the treatment group was gradually decreased, and the expression of GSK3-2 protein in the treatment group was significantly decreased, and the time point of the group was weak, 1d, 3d, 7d and 14d was significantly different from the corresponding time point of the model group (P0.05) (5) RT-PCR. The expression of Axin mRNA in the model group was gradually decreased with the increase of time, and the expression of Axin mRNA in the treatment group was less than that of the model group. The results showed that the expression of LEF1mRNA in the model group increased with the expression of 1 d after the ischemia-reperfusion, and the expression of LEF1mRNA in the treatment group was higher than that of the model group. The expression of LEF1mRNA in the treatment group was higher than that of the model group, among which the time-point ratio of the third day, the 7th day, the 14th day and the model group was statistically significant (P0. Conclusion: Ilex corylifolia has a significant effect on the neurological function of the rats after cerebral ischemia and reperfusion, and the mechanism may be by up-regulating the expression of the core molecule of Wnt pathway and promoting the entry of the antigen-cattenin, down-regulation of the inhibitory factor GSK3, Ax. in expression, the transcription of the downstream target gene LEF1 is activated, thereby activating the Wnt signaling pathway, promoting nerve regeneration, protecting,
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2014
【分類號】：R743.31

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