肾虚痰蒙清窍与老年性痴呆的关系及补肾化痰开窍法作用的研究

发布时间：2018-05-18 05:37

本文选题：老年性痴呆 + 泛素　；参考：《湖北中医药大学》2016年博士论文

【摘要】：目的:补肾化痰开窍法是中医学的重要治法之一,在老年性痴呆的防治过程中起着重要作用。本文系统回顾和总结了古代医家对老年性痴呆的认识,认为此病的基本病机是痰蒙清窍,肾元亏虚,属本虚标实之证。从肾虚痰蒙清窍理论探讨老年性痴呆的发病机理和治疗法则,本课题以APP/PS1双转基因小鼠作为老年性痴呆动物模型,观察补肾化痰开窍法对小鼠学习记忆水平、脑组织病理形态和泛素-蛋白酶体通路相的影响,探讨补肾化痰开窍法对该模型的干预作用和作用机理,旨在为补肾化痰开窍法防治老年病的研究提供理论基础和实验依据。方法:1.理论探讨:整理与中医补肾化痰开窍法有关的古今文献,分析补肾化痰开窍法的概念、内涵,探讨补肾化痰开窍法的理论基础,及其防治老年性痴呆的理论依据。2.以APP/PS1双转基因小鼠为老年性痴呆动物模型,泛素-蛋白酶体途径为研究靶点,研究补肾化痰开窍法对老年性痴呆的作用及其机制。观察跳台测试、Morris水迷宫测试对小鼠为行为学影响,检测小鼠的学习记忆能力;HE染色、免疫组织化学染色法观测小鼠海马组织泛素-蛋白酶体途径病理学变化,用Western Blot法检测小鼠海马组织泛素-蛋白酶体途径主要蛋白的含量;以Real Time-PCR检测小鼠海马组织泛素m RNA的表达。结果:1.Morris水迷宫测试结果:空白组比较,模型组小鼠发现平台时间明显延长(P0.01),游泳总路程明显增加(P0.01);与模型组比较,各组小鼠发现平台时间均缩短(P0.05),游泳总路程均减少(P0.05);各给药组与多奈哌齐组比较各组小鼠发现平台时间未见明显差异(P0.05),游泳总路程未见明显差异(P0.05)。益智温胆组与菖蒲益智丸组比较,两组小鼠发现平台时间未见明显差异(P0.05),游泳总路程未见明显差异(P0.05)。2.跳台测试实验结果给药90天后,与空白组比较,模型组小鼠的在跳台中错误次数明显增加(P0.01),潜伏期明显缩短(P0.01)。与模型组比较,多奈哌齐组和其它各组小鼠的出错次数明显减少(P0.01),多奈哌齐组、益智温胆组和菖蒲益智丸组小鼠的潜伏期明显延长(P0.01)。与多奈哌齐组比较,益智温胆组和菖蒲益智丸组小鼠的出错次数和潜伏期无明显差别(P0.05);补肾组化痰组出错次数和潜伏期有所差别(P0.05),化痰组潜伏期有所差别(P0.05)。3.HE染色模型组主要表现为较明显的脑神经元变性、可见到神经纤维缠结和淀粉样蛋白沉积斑块。多奈哌齐组和补肾组对模型转基因小鼠海马病理形态影响不明显,多奈哌齐组仅仅可以观察到神经元数量变化,以及细胞结构排列较为整齐,补肾组神经元形态排列较为整齐,结构较多奈哌齐组完整。益智温胆组、菖蒲益智丸组、化痰组对神经元的保护,空泡变性以及细胞核增大也有一定好转,神经细胞排列以及细胞形态明显较好,神经纤维缠结不明显。4.免疫组化泛素阳性免疫产物定位于海马区神经元细胞浆内。免疫组化结果显示模型组、多奈哌齐组切片内海马CA1区可见大量泛素,补肾组海马CA1区也可见到有部分泛素的聚集;益智温胆组与菖蒲益智丸组相差不多,二者最少。空白组小鼠海马CA1区灰度值明显较低(P0.01);而多奈哌齐组与其相差无几无明显差异(P0.05);益智温胆组、补肾组、化痰组与菖蒲益智丸组小鼠海马CA1区灰度值与其有明显差异(P0.01)。5.小鼠海马组织Western Blot实验结果APP/PS1双转基因小鼠海马组织泛素抗体蛋白质印迹检测结果表示:与空白组比较,模型组小鼠海马组织泛素相对表达量有显著性差别(P0.01)。与模型组比较,多奈哌齐组海马组织泛素相对表达量未见显著性差异(P0.05);模型组较其它各组均显示表达量增加。6.小鼠海马组织Real Time-PCR实验结果与空白组比较,模型组泛素m RNA表达明显上调,益智温胆组、补肾组、化痰组、菖蒲益智丸组泛素m RNA均有不同程度的上调。与模型组比较,多奈哌齐组、益智温胆组、补肾组、化痰组、菖蒲益智丸组泛素m RNA均有不同程度的下调。结论:1.肾虚痰蒙清窍是老年性痴呆的基本病机,补肾化痰开窍法能够有效的防治老年性痴呆。2.补肾化痰开窍法对APP/PS1双转基因小鼠的学习记忆能力具有改善作用。3.补肾化痰开窍法能有效的改善动物海马泛素化通路功能,
[Abstract]:Objective: the method of invigorating the kidney and eliminating phlegm and opening the orifices is one of the important treatment methods of traditional Chinese medicine and plays an important role in the prevention and treatment of Alzheimer's disease. This paper systematically reviews and summarizes the understanding of the Alzheimer's disease by ancient doctors. It is believed that the basic pathogenesis of this disease is phlegm, Mongolia, deficiency of the kidney and the deficiency of the kidney. The pathogenesis and treatment rules of Alzheimer's disease were treated with APP/PS1 double transgenic mice as the animal model of Alzheimer's disease. The effect of tonifying kidney and eliminating phlegm on the learning and memory level, the pathological morphology of brain tissue and the effect of ubiquitin proteasome pathway were observed. The effect and effect of the method of invigorating kidney and removing phlegm and opening the orifices on the model were discussed. The mechanism is designed to provide theoretical basis and experimental basis for the study on the prevention and treatment of senile disease by Tonifying the kidney and eliminating phlegm and opening the orifices. Method: 1. theoretical discussion: to organize the ancient and modern literature related to the method of invigorating the kidney and chemical phlegm, analyzing the concept and connotation of the method of invigorating the kidney and removing phlegm and opening the orifices, discussing the theoretical basis of the method of invigorating the kidney and resolving phlegm, and the theory of the prevention and treatment of senile dementia. According to.2., APP/PS1 double transgenic mice were used as the animal model of Alzheimer's disease, and the ubiquitin proteasome pathway was used as the target to study the effect and mechanism of kidney tonifying and phlegm orifices on Alzheimer's disease. The effect of Morris water maze test on behavioral learning in mice was observed, and the learning and memory ability of mice was detected, HE staining and immune tissue were observed. The pathological changes of ubiquitin proteasome pathway in hippocampal tissues of mice were observed by chemical staining. The content of the main proteins of ubiquitin proteasome pathway in hippocampal tissues of mice was detected by Western Blot method; the expression of ubiquitin m RNA in hippocampus of mice was detected by Real Time-PCR. Results: the results of 1.Morris water maze test: blank group comparison, model group mice It was found that the time of the platform was prolonged (P0.01) and the total distance of swimming was significantly increased (P0.01). Compared with the model group, the mice found the platform time shortened (P0.05) and the total swimming course decreased (P0.05). There was no significant difference (P0.05) between the group and the donepezil group (P0), and there was no significant difference in the total swimming course (P0). .05). Compared with the Acorus Yizi group, the two groups of mice found no significant difference (P0.05), and there was no significant difference in the total distance between the two groups (P0.05). After 90 days of the test, the number of errors in the model group was significantly increased (P0.01) and the incubation period was significantly shorter than that in the blank group (P0.01). In the model group, the number of errors in the donepezil group and the other mice decreased significantly (P0.01). The latency of the mice in the donepezil group, the Yizi Wendy group and the calamus Yizi pill group were obviously prolonged (P0.01). There was no significant difference between the Yizi Wendy group and the calamus Yizi pill group (P0.05). There was a difference in the frequency of error and incubation period in the kidney group (P0.05), and the latency of the phlegm group was different (P0.05) in the.3.HE staining model group, the most obvious degeneration of the brain neurons, the neurofibrillary tangles and amyloid plaques, and the effect of donepezil group and kidney supplement on the hippocampus pathological morphology of model transgenic mice was not affected. It was obvious that the number of neurons in the donepezil group could only be observed, and the structure of the cells was neatly arranged. The structure of the tonifying group was more neat and the structure was more complete. The protection of the neurons, the vacuoles degeneration and the nucleus enlargement of the group of Yizi wendyen, the calamus Yizi pill group and the phlegm group were also improved. The cell morphology and cell morphology were better, and the neurofibrillary tangles were not obvious in the hippocampal neuronal cytoplasm. The immunohistochemical results showed that in the model group, a large number of ubiquitin was found in the hippocampal CA1 area in the donepezil section, and some ubiquitin aggregation was found in the hippocampus CA1 area of the tonifying kidney group. The difference between the Yizi Wendy group and the calamus Yizi pill group was not much, the two was the least. The gray value of the hippocampal CA1 area in the blank group was significantly lower (P0.01), but there was no significant difference between the donepezil group and the group of donepezil (P0.05); the gray value of the hippocampus CA1 area of the rats of the Yizi Wendan group, the kidney tonifying group, the phlegm group and the calamus Yizi pill group was significantly different from that of the.5. mice (P0.01). Western Blot test results of hippocampal tissue in the hippocampus of APP/PS1 double transgenic mice showed that: compared with the blank group, there was a significant difference in the relative expression of ubiquitin in the hippocampus of the model group (P0.01). Compared with the model group, the relative expression of ubiquitin in the hippocampus of donepezil group was not significant difference. Compared with the other groups, the model group showed an increase in the expression of Real Time-PCR in the hippocampus of.6. mice compared with the blank group. The expression of ubiquitin m RNA in the model group was obviously up-regulated, and the level of ubiquitin m RNA in the Yizi Wendan group, the tonifying group, the phlegm group and the Acorus Yizi pill group were up to be up. Compared with the model group, the donepezil group and the beneficial intelligence were improved. The ubiquitin m RNA in the group of warm gallbladder, kidney tonifying, phlegm and Yizi Yizhi group have different downgrades. Conclusion: 1. the kidney deficiency and phlegm Mongolia clear orifices are the basic pathogenesis of senile dementia, the method of invigorating the kidney and eliminating phlegm and opening the orifices can effectively prevent and control the.2. invigorating phlegm and the orifices of the senile dementia, which can improve the learning and memory ability of the APP/PS1 double transfer gene mice,.3. Tonifying kidney, resolving phlegm and inducing resuscitation can effectively improve the function of hippocampus ubiquitination pathway in animals.
【学位授予单位】：湖北中医药大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：R277.7

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