益气活血法和补肾生髓法对脑缺血再灌注大鼠海马及皮质GSK3α、Sfrp1和Wnt9a蛋白表达的影响

发布时间：2018-03-25 14:12

本文选题：脑缺血　切入点：益气活血法　出处：《安徽中医药大学》2016年硕士论文

【摘要】：1目的本课题以中医学“肾精-脑髓”和“气血-血瘀”相关理论为依据,将Wnt/-catenin信号调控做为研究切入点,采用MCAO/R动物模型,主要通过益气活血法代表方脑络欣通方和补肾生髓法代表方补肾生髓方,对脑缺血再灌注大鼠进行干预,观察模型大鼠缺血侧海马及额顶叶皮质区GSK3α、Sfrp1和Wnt9a蛋白表达的影响,最后通过实验结果和相关实验数据,从分子生物学角度对益气活血法和补肾生髓法治疗缺血性中风的机制和原理进行阐述和分析。2方法首先选择健康Sprague-Dawley(SD)的雄性大鼠,总共56只,月龄4个月左右,体重在280-320g之间。随机将此56只实验大鼠分为4组,分别记为假手术组、模型组、益气活血法组和补肾生髓法组,每组14只,并为每组大鼠进行标记,将每组标记的大鼠再随机分为2组,记为3d、7d组,每组7只。采用改良线栓法建立局灶性右侧脑缺血大鼠模型,同时将益气活血法代表方脑络欣通方以及补肾生髓法代表方补肾生髓方,取中等剂量:8.54g/kg、12.87g/kg,对大鼠进行灌胃给药。益气活血法组和补肾生髓法组在复制MCAO/R模型前0.5h灌胃给药1次,以后于每天上午9:00和下午4:00分别灌胃一次,持续给药3d、7d,假手术组和模型组给予同等剂量的生理盐水灌胃。选取大鼠新鲜缺血侧海马及额顶叶皮质,应用Western-Blot技术检测GSK3α、Sfrp1和Wnt9a蛋白表达并加以分析,扫描显色后的底片,运用专业软件Image J对图像进行灰度分析,SPSS20.0处理所得数据,所得结果采用VISIO 2007软件进行图表绘制。3结果Western Blot结果显示:GSK3α条带位于46/51kda处,Sfrp1条带位于33kda处,Wnt9a条带位于37kda处,Actin条带位于42/44kda处。益气活血法3d组和补肾生髓法3d组对脑缺血再灌注大鼠缺血侧海马区GSK3α、Sfrp1和Wnt9a蛋白表达的影响:与假手术组比较,模型组三种蛋白的相对表达量均明显升高(P0.01);与模型组比较,益气活血法组和补肾生髓法组三种蛋白表达均明显降低(P0.01);与补肾生髓法组比较,益气活血法组三种蛋白表达均明显降低(P0.01)。额顶叶皮质区与海马区结果一致。益气活血法7d组和补肾生髓法7d组对脑缺血再灌注大鼠缺血侧海马区GSK3α、Sfrp1和Wnt9a蛋白表达的影响:与假手术组比较,模型组三种蛋白表达均明显升高(P0.01);与模型组比较,益气活血法组和补肾生髓法组三种蛋白表达均明显降低(P0.01);两种复方无显著差异(P0.05)。额顶叶皮质区与海马区结果一致。4结论两种中药复方能通过抑制缺血侧海马及皮质GSK3α、Sfrp1和Wnt9a蛋白表达,调节Wnt/-catenin信号通路,促进局灶性脑缺血再灌注损伤脑组织修复。
[Abstract]:Objective based on the theories of "kidney essence and brain marrow" and "qi, blood and blood stasis" in traditional Chinese medicine (TCM), the Wnt/-catenin signal regulation was taken as the starting point and the animal model of MCAO/R was used. The effects of GSK3 伪 -Sfrp1 and Wnt9a protein expression in hippocampus and frontal parietal cortex of model rats were observed by invigorating qi and activating blood circulation method on behalf of Nao Luo Xin Tong Fang and Bushen Shengmai Fang on behalf of Bushen Shengmai recipe, and Bushen Shengmai Fang on behalf of Nao Luo Xin Tong Fang and Bushen Sheng Mei Fang on behalf of supplementing Qi and activating Blood Circulation. Finally, through the experimental results and related experimental data, from the molecular biological point of view, the mechanism and principle of Yiqi Huoxue method and Bushen Shengmai method in treating ischemic apoplexy were expounded and analyzed. Firstly, the healthy Sprague-Dawley SD male rats were selected, with 56 rats in total. The 56 experimental rats were randomly divided into four groups: sham operation group, model group, invigorating qi and activating blood circulation group and tonifying kidney and generating marrow group, 14 rats in each group. Each group of labeled rats was randomly divided into 2 groups, which were divided into 3 days and 7 days group with 7 rats in each group. The model of focal right cerebral ischemia was established by modified thread occlusion method. At the same time, tonifying Qi and activating Blood Circulation method on behalf of Nao Luo Xin Tong Fang and Bushen Shengli method on behalf of Bushen Shengmai recipe were taken at a moderate dose of 8.54 g / kg or 12.87 g / kg. The rats in the Yiqi Huoxue method group and Bushen Shengmai method group were given orally once 0.5 hours before the MCAO/R model was established. The rats in sham operation group and model group were given the same dose of normal saline at 9:00 and 4:00 for 3 days and 7 days respectively. The fresh ischemic hippocampus and frontal parietal cortex of rats were selected. The expression and analysis of GSK3 伪 -Sfrp1 and Wnt9a protein were detected by Western-Blot technique. The negative films were scanned and processed by SPSS 20.0 with the professional software Image J. The results were drawn by VISIO 2007 software. 3 results Western Blot results showed that the Western Blot showed that the Sfrp1 band was located at the 46/51kda, the Wnt9a band was located at the 37kda, the actin band was located at the 42/44kda, the 3d group of supplementing qi and activating blood circulation method and the 3d group of the method of reinforcing kidney and generating marrow. Effects of GSK3 伪 -Sfrp1 and Wnt9a protein expression in the hippocampus of ischemic rats after cerebral ischemia-reperfusion: compared with sham-operated group, Compared with the model group, the relative expression of three kinds of proteins in the model group was significantly higher than that in the model group, and compared with the model group, the expression of the three proteins in the Yiqi Huoxue group and the tonifying kidney group was significantly lower than that in the Bushen group. The expression of three kinds of protein in Yiqi and Huoxue treatment group was significantly decreased, and the results of frontal parietal cortex and hippocampus were consistent. The expression of GSK3 伪 Sfrp1 and Wnt9a protein in the ischemic hippocampus of rats with cerebral ischemia and reperfusion was observed in 7 days group of supplementing qi and activating blood circulation method and 7 days group of tonifying kidney and generating marrow method. Effects: compared with the sham-operated group, The expression of three proteins in the model group was significantly higher than that in the model group. The expression of three kinds of protein was significantly decreased in the two groups, and there was no significant difference between the two groups. The results of frontal parietal cortex and hippocampus were consistent with that of hippocampus. Conclusion the two Chinese herbs can inhibit the ischemic hippocampus and the skin of the ischemic side. The expression of Sfrp1 and Wnt9a proteins in the cytoplasm of GSK3 伪 -Sfrp1, Regulate Wnt/-catenin signal pathway and promote brain tissue repair after focal cerebral ischemia reperfusion injury.
【学位授予单位】：安徽中医药大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：R255.2

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