毛冬青甲素对脑缺血后经典Wnt通路的调控作用与神经再生

发布时间：2019-06-19 20:57

【摘要】：目的：本研究拟通过观察毛冬青甲素（IlexoninA，IA）对大鼠脑缺血再灌注后Wnt信号通路的调控作用和神经再生的情况，探讨IA促进神经元再生及脑保护作用的可能机制。方法：SD大鼠(雄性)随机分为正常组、假手术组（手术过程同模型组，只插入线栓不阻塞大脑中动脉）、模型组、IA治疗组，各组又分为再灌注1d、3d、7d、14d四个亚组，每组各9只。用线栓阻塞大脑中动脉制作大鼠局灶性脑缺血再灌注模型，通过Longa法对大鼠的神经功能进行评分；通过脑组织TTC染色观察脑缺血再灌注损伤后的脑梗死情况；通过免疫荧光双标的方法观察缺血周边组织Brdu/nestin、Brdu/NueN双标阳性细胞数；通过免疫荧光、western bolt、RT-PCR检测经典Wnt信号通路相关因子β-catenin、GSK3β、Axin、LEF-1的表达情况。结果：（1）神经功能缺损的症状于脑缺血再灌注后第1d明显可见，症状随时间的推移有所改善。神经功能缺损的评分治疗组较模型组低，治疗组第3d和7d与模型组对应的时间点相比差异有显著性意义（P0.05）。（2）缺血周边组织细胞情况：模型组和治疗组Brdu/nestin、Brdu/NeuN双标阳性细胞于再灌注后第3d表达明显增多，第7d开始下降，到第14d时仍有少量表达，3d表达最多；其中治疗组Brdu/nestin、Brdu/NeuN双标阳性细胞于缺血再灌注后第3d、7d与模型组相应时间点比较有显著性差异（P0.05）（3）缺血周边组织β-catenin的表达情况：模型组入核β-catenin阳性细胞数量再灌注后第1d开始表达，第3d表达达高峰，第7d开始下降，第14d仍有表达；治疗组各时段入核β-catenin阳性细胞表达明显多于模型组，其中第3d、7d与模型组相对应的时间点比差异有统计学意义（P0.05）。（4）免疫蛋白印迹：①β-catenin蛋白表达情况：模型组β-catenin蛋白表达于缺血再灌注后1d开始增多，3d表达最多，其条带密度明显增高，而后逐渐下降，，至14d仍有表达，治疗组β-catenin蛋白较模型组明显增高，1d、3d、7d与模型组相应时间点比较有显著性差异（P0.05）。β-catenin蛋白表达情况所得结果与免疫荧光结果相吻合。②GSK3β蛋白表达情况：模型组GSK3β蛋白随时间的延长，表达逐渐减弱，治疗组GSK3β蛋白各时段表达明显减少，条带较淡，1d、3d、7d、14d与模型组相应时间点比较有显著性差异（P0.05）（5）RT-PCR法检测：①Axin mRNA表达情况：模型组Axin mRNA随时间的延长表达逐渐减弱，治疗组Axin mRNA表达较模型组各时段有所减少，条带较淡，其中3d、7d与模型组相应时间点比较有显著性差异（P0.05）②LEF1mRNA表达情况：模型组LEF1mRNA于缺血再灌注后1d表达增多，3d表达最多，而后逐渐下降，至14d仍有表达，治疗组LEF1mRNA表达较模型组高，其中第3d、7d、14d与模型组相对应时间点比差异有统计学意义（P0.05）. 结论：毛冬青甲素对大鼠脑缺血再灌注后的神经功能有明显改善作用，其机制可能是通过上调Wnt通路核心分子β-catenin的表达及促进β-catenin蛋白入核，下调抑制性因子GSK3β、Axin的表达，激活下游靶基因LEF1的转录，从而激活Wnt信号通路，促进神经再生，保护受损脑组织。
[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,
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R743.31

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