基于Aβ血脑屏障清除学说探讨电针干预AD模型脑LRP1及其血管调节基因表达的影响

发布时间：2018-05-11 08:12

本文选题：阿尔茨海默病 + 血脑屏障　；参考：《北京中医药大学》2017年博士论文

【摘要】：1研究目的阿尔茨海默病(Alzheimer' s Disease,AD)又称老年痴呆,是一种以进行性认知障碍和记忆能力损害为主的中枢神经系统退变性疾病。阿尔茨海默病是当前公共健康所面临的重大问题,已被确定为中枢神经系统损伤领域研究的重点疾病。因此研究老年性痴呆及其相关疾病已成为了刻不容缓的迫切任务,成为国内外医学研究的重点课题之一。临床研究显示,针灸可以改善AD患者的精神行为学状态和认知功能。电针是一种可以针对多种疾病简单有效的治疗手段,许多的研究和证据表明针刺可以有效地改善AD,但其机制尚不明确,需要进一步探讨。Aβ级联假说认为β淀粉样蛋白(β Amyloid protein,Aβ)的生成与清除失衡是其沉积于脑组织继而形成淀粉样斑块的原因,其可能具有的神经毒性作用引发神经元变性,导致AD症状的产生。目前,也已经有许多团队从多个角度研究并证明其确实存在神经毒性,Aβ在神经的退化和凋亡过程中扮演着十分重要的角色。本课题组以电针"百会、涌泉"为治疗手段,研究电针对不同月龄APP/PS1转基因小鼠行为学的影响,探讨电针治疗阿尔兹海默病的最佳干预时间;基于A0的血脑屏障清除途径,观察电针对的APP/PS1转基因AD模型脑内和血清Aβ1-40和Aβ 1-42的表达,探讨电针是否通过调节APP/PS1双转基因鼠低密度脂蛋白受体蛋白-1(LRP1)相关基因的表达,促进海马内β淀粉样蛋白清除,探讨电针治疗阿尔兹海默病的机制。2研究方法首先,将4、6、9月龄APP/PS1双转基因小鼠各20只,随机分为模型组、电针治疗组,以相同月龄C57BL/6野生鼠各10只作为正常对照组;电针干预6周后,采用Morris水迷宫进行空间记忆行为学测试,观察各月龄各组行为学的变化。之后,将6月龄APP/PS1双转基因小鼠64只,随机分为模型组、电针治疗组,以C57BL/6野生鼠32只作为正常对照组;采用Morris水迷宫进行空间记忆行为学测试;以免疫组化法观察脑内A β 1-42及LRP1的表达,激光共聚焦观察A β 1-42及LRP1的共表达情况,二次过筛法取微血管段观察LRP1表达;ELISA法检测皮层、海马和血清Aβ1-42,Western Blotting检测海马LRP1表达水平,免疫组化和RT-PCR法用来检测海马MEOX2、MYOCD、SREBP2 表达。3研究结果3.1 Morris水迷宫检测显示:三个月龄逃避潜伏期电针组相对于模型组都有下降趋势。重复测量统计显示:5月龄组间效应方差分析无统计学意义(P0.05),7月龄组间效应方差分析、时间因素及交互作用均有显著有明显差异(P0.01);10月龄组间效应方差分析、时间因素、交互作用均有统计学意义(P0.05)三个月龄穿越平台次数及平台象限游泳路程,模型组均明显低于正常对照组(P0.05),针刺组高于模型组。3.2 7月龄Morris水迷宫检测显示:模型组与正常对照组比较逃避潜伏时增加、空间探索实验穿越平台次数、平台象限游泳路程明显减少(P0.05,P0.01),电针治疗组逃避潜伏时明显减少(P0.05);激光共聚焦和免疫组化显示:模型组有明显的致密性老年斑沉积,针刺组相对减少;电针组LRP1在血管周围表达相对增多。3.3 ELISA检测皮层、海马和血清A β 1-40、Aβ1-42电针治疗组较模型组都明显降低(P0.01)。WB和ELISA结果显示LRP1表达水平,模型组低于正常组(P0.01),而电针组高于模型组。3.4PCR结果显示MEOX2表达水平,模型组低于正常组(P0.01),而电针组高于模型组(P0.01);MYOCD、SREBP2的表达,模型组高于正常组(P0.01),而电针组低于模型组(P0.01);与免疫组化表达结果相一致。4结论4.1电针可以改善APP/PS1双转基因鼠的学习记忆能力,6月龄可能是电针早期干预AD的最佳治疗介入点,但仍需进一步实验机制研究讨论。4.2电针可降低皮层、海马和血清中A β水平,且电针组具有神经毒性的A β 1-42的水平降低较明显,这可能是电针治疗痴呆的作用途径之一。4.3电针可以改善APP/PS1双转基因小鼠的学习记忆能力,降低脑内Aβ的表达,其机制可能与A β转运受体LRP1水平升高有关。4.4电针上调MEOX2,降低MYOCD、SREBP2来干预A β转运受体LRP1相关,这可能是电针干预AD血脑屏障清除A β治疗AD的可能机制。
[Abstract]:1 Alzheimer's Disease (AD), also known as Alzheimer's disease, is a central nervous system degenerative disease characterized by progressive cognitive impairment and memory impairment. Alzheimer's disease is a major problem facing public health, and has been identified as a key disease in the field of central nervous system injury. Therefore, the study of Alzheimer's disease and related diseases has become an urgent and urgent task, which has become one of the key topics in medical research both at home and abroad. Clinical studies have shown that acupuncture can improve the mental and behavioral state and cognitive function of AD patients. Research and evidence suggest that acupuncture can effectively improve AD, but its mechanism is not clear. It is necessary to further explore the.A beta cascade hypothesis that the formation and clearance of beta amyloid (beta Amyloid protein, A beta) is the cause of its deposition of amyloid plaques in the brain tissue, and its possible neurotoxicity triggering neuronal degeneration. Sex, leading to the emergence of AD symptoms. At present, many teams have studied and proved that they do exist neurotoxicity from multiple angles. A beta plays a very important role in the process of neural degeneration and apoptosis. In this group, the behavior of Electroacupuncture on APP/PS1 transgenic mice of different months of age is studied by Electroacupuncture "Baihui, Yongquan" as the treatment method. To explore the best intervention time of electroacupuncture in treating Alzheimer's disease, the expression of A beta 1-40 and A beta 1-42 in the brain and serum of the APP/PS1 transgenic AD model based on the Electroacupuncture of A0 based on the blood brain barrier clearance pathway, and to explore whether the electroacupuncture can be used to regulate the -1 (LRP1) related genes of the low density lipoprotein receptor protein receptor protein of the APP/PS1 double transgenic mice. To promote the clearance of amyloid protein in the hippocampus and explore the mechanism.2 research method of electroacupuncture treatment of Alzheimer's disease first, 20 4,6,9 month old APP/PS1 double transgenic mice were randomly divided into model group, electroacupuncture treatment group, 10 of the same month old C57BL/6 wild rats as the normal control group; after 6 weeks of Electroacupuncture intervention, the Morris water maze was used. 6 month old APP/PS1 double transgenic mice were randomly divided into model group, electroacupuncture treatment group and 32 C57BL/6 wild rats as normal control group, and Morris water maze was used to conduct spatial memory behavior test, and A beta 1-4 in brain was observed by immunohistochemical method. 2 and LRP1 expression, confocal laser confocal observation of the co expression of A beta 1-42 and LRP1, two times of screening microvascular segment to observe the expression of LRP1, ELISA method to detect the cortex, hippocampus and serum A beta 1-42, Western Blotting to detect the expression level of hippocampus LRP1, immunohistochemistry and RT-PCR method used to test the hippocampus MEOX2, MYOCD, 3.1 expresses the result of MYOCD. The is water maze test showed that the three month old escape incubation group had a downward trend relative to the model group. The repeated measurements showed that there was no significant difference in variance analysis between the 5 month old groups (P0.05), and there were significant differences between the 7 month old groups of effect variance analysis, time factors and interaction (P0.01), and the variance of the effect between the 10 month old groups. The analysis, time and interaction were statistically significant (P0.05) three months of age crossing platform and platform quadrant swimming distance, the model group was significantly lower than the normal control group (P0.05), and the acupuncture group was higher than the model group.3.2 7 month old Morris water maze test. The model group was compared with the normal control group, and the space exploration was higher than the normal control group. The number of traversing platform, the platform quadrant swimming path decreased significantly (P0.05, P0.01), the electroacupuncture treatment group decreased obviously (P0.05). Laser confocal and immunohistochemical staining showed that the model group had obvious densification of the senile plaque and the relative decrease in the acupuncture group; the expression of LRP1 in the electroacupuncture group was relatively increased by.3.3 ELISA to detect the cortex and the sea. The A beta 1-40 and A beta 1-42 electroacupuncture group were significantly lower than the model group (P0.01) and the results of.WB and ELISA showed LRP1 expression level, the model group was lower than the normal group (P0.01), and the electroacupuncture group was higher than the model group, and the.3.4PCR results showed the MEOX2 expression level, the model group was lower than the normal group (P0.01), and the electroacupuncture group was higher than the model group (P0.01); MYOCD, the table of the.3.4PCR group was higher than the model group. The model group was higher than the normal group (P0.01), but the electroacupuncture group was lower than the model group (P0.01), and the.4 conclusion 4.1 electroacupuncture could improve the learning and memory ability of the APP/PS1 double transgenic mice. The 6 month old may be the best intervention point for the early intervention of AD by electroacupuncture, but the further experimental mechanism was still needed to discuss the reduction of.4.2 electroacupuncture. The level of A beta in the lower cortex, hippocampus and serum, and the level of neurotoxic A beta 1-42 in the electroacupuncture group decreased significantly. This may be one of the ways of treating dementia by electroacupuncture..4.3 electroacupuncture can improve the learning and memory ability of APP/PS1 double transgenic mice and reduce the expression of A beta in the brain. The mechanism may be associated with the LRP1 level of A beta transporter LRP1. High related.4.4 electroacupuncture up regulation of MEOX2, reduced MYOCD, and SREBP2 to interfere with LRP1 related A beta transporter receptor, which may be a possible mechanism for the interference of electroacupuncture in the AD blood-brain barrier to eliminate A beta in the treatment of AD.

【学位授予单位】：北京中医药大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R245;R-332

【参考文献】