针灸对AD模型大鼠海马区Aβ内化相关蛋白酶的影响研究

发布时间：2018-03-03 07:06

本文选题：阿尔茨海默病　切入点：星形胶质细胞　出处：《湖北中医药大学》2017年硕士论文　论文类型：学位论文

【摘要】：目的:老年斑是阿尔茨海默病特征性病理改变之一,β淀粉样蛋白是老年斑的核心物质,因此减少Aβ的过量生成或加速其内化,减少Aβ的聚集、沉降,是改善阿尔茨海默病病理进程的重要途径之一。根据目前已经发表的研究文献,有关Aβ的生成途径方面的研究已有了一定深度的探索,而有关Aβ内化方面的研究还需要更进一步的深入。本课题从Aβ降解的方面进行研究,选取脂蛋白酯酶、抗凝乳蛋白酶α、载脂蛋白E作为研究指标,通过观察针灸百会和肾俞穴对星形胶质细胞的激活,及此三种蛋白酶与Aβ关系,探讨“益肾调督”针灸法治疗阿尔茨海默病的可能作用机制。方法:选用Wistar大鼠、SPF级、雄性、体重(360±20)g,造模前所有大鼠经Morris水迷宫筛选,将符合条件的60只Wistar大鼠随机分为正常组、假手术组、模型组、治疗组。模型组用微量注射法向双侧海马区各注射5μL寡聚态Aβ1-42;假手术组用同样方法在双侧海马区注射与Aβ1-42等量的生理盐水;治疗组在造模成功的基础上选取百会穴和肾俞穴进行常规针刺(针具规格:长25mm直径0.35mm毫针),选用细艾条(半径约0.25mm)在大鼠腧穴上方2~3cm处进行温和灸,每穴15min;正常组不做任何处理,常规饲养。每日1次,6天为1个疗程,共2个疗程,疗程间休息1日。针灸治疗结束后,各组选取10只进行指标检测:(1)采用Morris水迷宫检测大鼠的学习记忆能力;(2)采用ELISA法检测血清中Aβ1-42的含量;(3)采用免疫组化法检测大鼠海马区GFAP蛋白的表达水平;(4)采用Western-Blot法检测大鼠海马区GFAP和Aβ内化相关蛋白酶ACT、LPL、ApoE的表达。结果:两个疗程治疗结束后,(1)行为学观察:与模型组相比,治疗组大鼠跨越平台数增加(P0.01),而逃避潜伏期则缩短(P0.01)。(2)ELISA检测结果:与正常组相比,模型组Aβ1-42明显升高(P0.01),假手术组Aβ1-42未有明显差异(P0.05);与模型组相比,治疗组Aβ1-42明显降低(P0.05)。(3)免疫组化结果:和正常组相比,模型组GFAP表达升高,具有统计学差异(P0.01);和模型组相比,治疗组平均光密度值没有显著差异(P0.05);和正常组比较,治疗组GFAP平均光密度值具有显著差异(P0.05)。(4)Western-Blot检测结果:与正常组相比,模型组GFAP蛋白表达显著升高(P0.01),假手术组GFAP表达则未有明显差异(P0.05);与模型组相比,治疗组GFAP表达稍降低,无统计学差异(P0.05)。与正常组相比,模型组LPL、ApoE蛋白表达显著降低(P0.01),ACT表达显著升高(P0.01),假手术组LPL、ApoE、ACT表达则未有明显差异(P0.05);与模型组比较,治疗组LPL、ApoE表达显著升高(P0.05),ACT表达显著降低(P0.05)。结论:“益肾调督”针灸法可以通过上调大鼠海马区LPL和ApoE的表达,下调ACT的含量,促进Aβ的内化,减少β淀粉样蛋白的沉积,从而延缓或改善阿尔茨海默病的病理进程。
[Abstract]:Objective: senile plaque is one of the characteristic pathological changes of Alzheimer's disease, 尾 -amyloid protein is the core substance of senile plaque, so it reduces the excessive production of A 尾 or accelerates its internalization, reduces the aggregation and sedimentation of A 尾. It is one of the important ways to improve the pathological process of Alzheimer's disease. According to the published research literature, the research on the pathway of A 尾 production has been deeply explored. However, the study on A 尾 internalization needs further study. In this study, lipoprotein esterase, anticoagulase 伪 and apolipoprotein E were selected as the research indexes from the aspect of A 尾 degradation. By observing the activation of astrocytes at Baihui and Shenshu points of acupuncture and moxibustion, and the relationship between these three proteases and A 尾, the possible mechanism of acupuncture and moxibustion method of "tonifying kidney and regulating du" in treating Alzheimer's disease was discussed. All the rats were screened by Morris water maze before modeling. 60 Wistar rats were randomly divided into normal group, sham operation group and model group. In the model group, 5 渭 L oligomeric A 尾 1-42 was injected into the bilateral hippocampal area by microinjection, while in the sham operation group, the same amount of normal saline as A 尾 1-42 was injected in the bilateral hippocampal area. On the basis of successful modeling, the treatment group selected Baihui point and Shenshu point for routine acupuncture (needle size: 25mm in diameter: 0.35 mm in diameter), and selected fine moxa bar (radius about 0.25 mm) for mild moxibustion at 2cm above the acupoint of rats. Each acupoint 15 minutes; normal group do not do any treatment, routine feeding. Once a day for 6 days as a course of treatment, a total of 2 courses of treatment between the rest on 1st. After acupuncture treatment, 10 rats in each group were selected for index detection. (Morris water maze was used to detect the learning and memory ability of rats.) the content of A 尾 1-42 in serum was detected by ELISA method. The expression level of GFAP protein in hippocampal area of rats was detected by immunohistochemical method. Western-Blot method was used to detect the expression of GFAP and A 尾 -internalization-associated protease ACTLPLApoE in the hippocampus of rats. Results: after two courses of treatment, we observed the behavior: compared with the model group, In the treatment group, the number of cross platform increased, while the escape latency was shortened. The results of Elisa: compared with the normal group, A 尾 1-42 in the model group was significantly higher than that in the control group, and there was no significant difference in A 尾 1-42 between the sham operation group and the model group, while in the model group, there was no significant difference between the two groups, while in the model group, there was no significant difference between the two groups. Compared with the normal group, the expression of GFAP in the model group was significantly higher than that in the control group (P 0.01), and there was no significant difference in the mean optical density between the treatment group and the model group (P 0.05). The results of Western-Blot analysis showed that the expression of GFAP protein in the model group was significantly higher than that in the normal group, but there was no significant difference in the expression of GFAP in the sham operation group, and the expression of GFAP in the treatment group was slightly lower than that in the model group. Compared with the normal group, the expression of LPLApoE protein in the model group was significantly lower than that in the control group, and the expression of P0.01ACT in the model group was significantly higher than that in the sham operation group, but there was no significant difference in the expression of ACT between the model group and the model group, and there was no significant difference in the expression of ACT between the model group and the model group, and there was no significant difference in the expression of ACT between the model group and the model group. In the treatment group, the expression of LPLO ApoE increased significantly and the expression of P0.05ACT decreased significantly. Conclusion: the acupuncture and moxibustion method of "tonifying kidney and regulating du" can down-regulate the content of ACT, promote the internalization of A 尾 and reduce the deposition of 尾 -amyloid protein by up-regulating the expression of LPL and ApoE in hippocampus of rats. Thus delaying or improving the pathological process of Alzheimer's disease.
【学位授予单位】：湖北中医药大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R245;R-332

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