蓝莓提取物改善阿尔茨海默病认知功能障碍的实验研究

发布时间：2018-04-26 11:25

本文选题：蓝莓 + 阿尔茨海默病　；参考：《天津医科大学》2013年博士论文

【摘要】：目的 (1)评价APP/PS1双转基因AD小鼠模型效果,明确其发病进程和特点,为后续研究提供一种稳定的AD动物模型；(2)观察蓝莓提取物(Blueberry extracts, BB)干预对AD动物学习记忆损伤的作用,以及对Ap诱导海马神经元损伤的拮抗作用；(3)探讨BB上述作用的可能机制。方法 AD转基因小鼠模型的建立：由两对APP/PS1小鼠繁育扩种,所繁殖的小鼠在40日龄时取鼠尾组织提取DNA,通过PCR扩增后琼脂糖电泳检测目标基因筛选出阳性小鼠；在7月龄时,分别对阳性小鼠和阴性小鼠进行Morris水迷宫实验测试,评价小鼠是否出现学习记忆能力损伤,之后取脑组织及海马组织进行HE染色和刚果红染色,观察是否出现类似AD的典型病理改变。 BB改善AD学习记忆障碍的实验研究：(1)体外实验：培养新生大鼠海马原代神经元,设置正常对照组、Aβ组以及Ap+不同浓度BB组,以BB预孵育24h后,再以5μmol/L Aβ25-35处理24h模拟AD损伤,MTT法检测各组神经元活力,并确定BB干预的有效剂量;(2)体内试验：根据基因型检测结果,将小鼠分成阴性对照(CT,溶剂对照)组、模型(AD,溶剂对照)组和模型干预(AD+BB, BB150mg/kg-bw)组。在小鼠3月龄时,对各组施加相应干预,持续16周,观察其外观变化,记录摄食量,Morris水迷宫试验评价学习记忆能力；之后处死小鼠,称量小鼠主要脏器重量并计算脏器系数；对脑皮层及海马组织进行病理检查,并计数凋亡、坏死神经元数目。 BB改善AD学习记忆相关机制的探讨：分别从细胞水平和整体水平进行研究。实验分组及处理同上。采用实时荧光定量PCR (RT-PCR)检测MAPK/ERK信号通路分子ERK1/2、MEK2,甲基化酶DNMT1、DNMT3a和DNMT3b,组蛋白去乙酰化酶HDAC1、HDAC2、HDAC3,脑源性神经营养因子(BDNF)和泛素羧基末端水解酶(UCH-L1)的mRNA表达；采用Western blotting检测ERK1/2、MEK2、DBDNF和UCH-L1蛋白的表达；检测小鼠脑组织及血清抗氧化防御系统功能,包括MDA.SOD、还原型GSH和GSH-Px;采用电生理学方法检测小鼠海马CA1区长时程增强(LTP)。分别从抗氧化、细胞信号转导、表观遗传学及突触可塑性的角度探索蓝莓神经保护作用的可能机制。结果 AD转基因小鼠模型的建立：Morris水迷宫检测结果显示,至7月龄时,APP/PS1小鼠定向航行试验需要耗费较长时间才能到达平台,对训练成果记忆稳定和牢固程度较差；空间探索试验结果显示,APP/PS1小鼠在目标象限区域的穿越次数明显少于正常对照小鼠,出现明显的学习记忆能力损伤。HE染色显示,7月龄的APP/PS1小鼠脑组织及海马CA1、CA3区均出现明显的神经元凋亡、变性,神经元减少；刚果红染色表明,阳性小鼠7月龄时脑组织已出现p淀粉样蛋白沉积斑块。 BB改善AD学习记忆障碍的实验研究：(1)细胞实验显示,0.4μg/mlBB可以对抗Ap损伤,提高神经元活力,而4μg/ml及以上浓度的BB反而影响细胞生长,抑制细胞活力,并且随浓度的增高,细胞活力下降程度与BB浓度之间呈现剂量依赖关系。(2)动物实验结果表明,BB干预可减轻AD模型小鼠老化的部分症状和体征,明显缩短AD小鼠在定向航行试验中的潜伏时间,增加在空间探索试验中的目标区域穿越次数,抑制大脑萎缩,病理检查发现BB还能减轻AD模型小鼠脑组织神经元凋亡、坏死程度。 BB改善AD学习记忆相关机制的探讨：(1)体外实验：Ap损伤可明显上调神经元ERK1/2、MEK2及三种HDAC的mRNA和蛋白的表达,同时抑制BDNF、UCH-L1的mRNA及蛋白的表达；BB干预后神经元ERK1/2、MEK2及三种HDAC表达上调的趋势得到明显抑制,而BDNF、UCH-L1的mRNA和蛋白表达明显增加。(2)动物实验：与CT组小鼠相比,AD小鼠脑组织中氧化损伤明显,BB可显著提高GSH-Px活性,升高还原型GSH,降低MDA水平。RT-PCR和Western blotting检测结果与细胞实验一致,AD小鼠脑组织ERK1/2、 MEK2和三种HDAC表达明显增高,但是BDNF和UCH-L1的表达明显减少。BB干预可有效抑制ERK1/2和MEK2的过度激活,减少HDAC的表达,上调BDNF和UCH-L1的表达。电生理学检测也显示,BB可以显著增强AD小鼠海马CA1区LTP。体外及动物实验均未发现AD状态下以及实施蓝莓干预对甲基化酶的表达存在显著差异。结论 (1)该AD转基因小鼠模型繁殖阳性率较稳定,AD样病理及行为特征表现典型,是一种理想的AD动物模型；(2)BB具有拮抗Ap损伤、保护神经元以及改善AD转基因小鼠学习记忆能力损伤的作用,但是BB可能存在安全剂量范围,过高的BB浓度对细胞生长可能存在不良影响。(3)AD发病可能通过加剧脑组织的氧化损伤、诱导MAPK/ERK信号通路相关蛋白的过度表达和活化、改变某些表观遗传学特征、影响神经突触相关蛋白的表达,对脑功能造成进行性的损伤；而BB可以有效拮抗AD过程中的氧化损伤,调节MAPK/ERK信号蛋白的异常表达和活化,抑制表观遗传的异常改变,增强突触可塑性,从而发挥其神经保护功能。
[Abstract]:objective
(1) evaluate the effect of APP/PS1 double transgenic AD mouse model, clarify its pathogenesis and characteristics, provide a stable AD animal model for follow-up study, (2) observe the effect of blueberry extract (Blueberry extracts, BB) intervention on learning and memory damage of AD animals, and the antagonism of Ap to induce hippocampal neuron injury; (3) discuss the above of BB above. The possible mechanism of action.
Method
The establishment of AD transgenic mice model: two pairs of APP/PS1 mice were bred, and the mice were bred to extract DNA from the rat tail tissue at 40 days of age. The positive mice were screened by agarose electrophoresis after PCR amplification. At 7 month old, the Morris water maze test was carried out to the positive and negative mice to evaluate the mice. The impairment of learning and memory ability was found, then brain tissue and hippocampal tissue were stained with HE and Congo red, and the typical pathological changes of AD were observed.
BB to improve the learning and memory impairment of AD: (1) in vitro experiment: the primary neurons of the hippocampus were cultured in vitro. The normal control group, the normal control group, the A beta group and the BB group with different concentrations of Ap+ were used to incubate 24h with BB, and then the 24h AD injury was simulated with 5 u mol/L A beta 25-35, and the MTT method was used to determine the effective dose of the intervention. (2) In vivo test: according to the results of genotyping, the mice were divided into negative control (CT, solvent control) group, model (AD, solvent control) group and model intervention group (AD+BB, BB150mg/kg-bw). At 3 month old mice, corresponding intervention was applied to each group for 16 weeks, the appearance changes were observed, food intake was recorded, and Morris water maze test was used to evaluate learning and memory ability. The mice were killed, the weight of the main organs of the mice was weighed and the organ coefficient was calculated. The pathological examination of the cerebral cortex and hippocampus was carried out, and the number of apoptotic and necrotic neurons was counted.
BB to improve the mechanism of learning and memory of AD: study in cell level and overall level respectively. Experimental grouping and processing on the same. Use real-time fluorescence quantitative PCR (RT-PCR) to detect MAPK/ERK signaling pathway molecule ERK1/2, MEK2, methylation enzyme DNMT1, DNMT3a and DNMT3b, group of egg white deacetylase HDAC1, HDAC2, brain origin God operation MRNA expression of nutrient factor (BDNF) and ubiquitin carboxyl terminal hydrolase (UCH-L1), expression of ERK1/2, MEK2, DBDNF and UCH-L1 protein were detected by Western blotting, and the function of antioxidant defense system in mouse brain tissue and serum, including MDA.SOD, reductive GSH and GSH-Px, was detected by electrophysiologic method. LTP). The possible mechanisms of neuroprotective effects of blueberry were explored from the perspectives of antioxidant, cell signaling, epigenetics and synaptic plasticity.
Result
The establishment of AD transgenic mice model: the results of Morris water maze test showed that at 7 month old, the directional navigation test of APP/PS1 mice took a long time to reach the platform, and the memory stability and firmness of the training results were poor. The results of space exploration test showed that the number of crossing times of the APP/PS1 mice in the target quadrant area was significantly less. In normal control mice, obvious learning and memory impairment.HE staining showed that 7 month old APP/PS1 mice brain tissue and hippocampal CA1, CA3 area showed obvious neuronal apoptosis, degeneration, and neuron decrease; Congo red staining showed that the P amyloid plaques appeared in the brain tissue at 7 month old of the positive mice.
BB experimental studies on improving the learning and memory disorders of AD: (1) cell experiments showed that 0.4 mu g/mlBB could antagonism Ap damage and increase neuron vitality, while BB of 4 mu g/ml and above concentration affected cell growth and inhibited cell viability, and with the increase of concentration, the degree of cell vitality decreased with BB concentration in a dose dependence. (2) animal real The results showed that BB intervention could reduce partial symptoms and signs of aging in AD model mice, shorten the latency time of AD mice in directional navigation test, increase the number of target area crossing in the space exploration test and inhibit brain atrophy. Pathological examination found that BB could reduce the apoptosis and necrosis of brain tissue in AD model mice.
BB to improve the mechanism of AD learning and memory: (1) in vitro experiment: Ap damage can obviously increase the expression of mRNA and protein of neuron ERK1/2, MEK2 and three kinds of HDAC, and inhibit the expression of BDNF, UCH-L1 mRNA and protein. The expression of RNA and protein increased significantly. (2) animal experiments: compared with the CT mice, the oxidative damage in the brain tissue of AD mice was obvious. BB could significantly increase the activity of GSH-Px, increase the original GSH, reduce the MDA level.RT-PCR and Western blotting, and the AD mouse brain tissue ERK1/2, and three kinds of expressions were significantly increased. The expression of BDNF and UCH-L1 significantly reduced.BB intervention to effectively inhibit the overactivation of ERK1/2 and MEK2, reduce the expression of HDAC, and up regulate the expression of BDNF and UCH-L1. Electrophysiological detection also showed that BB could significantly enhance the LTP. in vitro and animal experiments in CA1 region of AD mice and the implementation of blueberry intervention against methylation enzymes. There were significant differences in expression.
conclusion
(1) the positive rate of AD transgenic mice was more stable, AD like pathological and behavioral characteristics were typical, and it was an ideal AD animal model. (2) BB has the effect of antagonizing Ap damage, protecting neurons and improving the learning and memory impairment of AD transgenic mice, but BB may have a safe dose range and high BB concentration to the cells. Growth may have adverse effects. (3) AD may induce the oxidative damage of brain tissue, induce overexpression and activation of MAPK/ERK signaling pathway related proteins, change some epigenetic characteristics, affect the expression of synapse related proteins, cause progressive injury to brain function, and BB can effectively antagonize the process of AD. Oxidative damage regulates the abnormal expression and activation of MAPK/ERK signal proteins, inhibits abnormal epigenetic changes, enhances synaptic plasticity, and thus exerts its neuroprotective function.

【学位授予单位】：天津医科大学
【学位级别】：博士
【学位授予年份】：2013
【分类号】：R285.5;R749.16

【参考文献】