本文選題：藍(lán)莓 + 阿爾茨海默病��； 參考：《天津醫(yī)科大學(xué)》2013年博士論文

【摘要】：目的 (1)評價APP/PS1雙轉(zhuǎn)基因AD小鼠模型效果,明確其發(fā)病進(jìn)程和特點,為后續(xù)研究提供一種穩(wěn)定的AD動物模型；(2)觀察藍(lán)莓提取物(Blueberry extracts, BB)干預(yù)對AD動物學(xué)習(xí)記憶損傷的作用,以及對Ap誘導(dǎo)海馬神經(jīng)元損傷的拮抗作用；(3)探討B(tài)B上述作用的可能機(jī)制。 方法 AD轉(zhuǎn)基因小鼠模型的建立：由兩對APP/PS1小鼠繁育擴(kuò)種,所繁殖的小鼠在40日齡時取鼠尾組織提取DNA,通過PCR擴(kuò)增后瓊脂糖電泳檢測目標(biāo)基因篩選出陽性小鼠；在7月齡時,分別對陽性小鼠和陰性小鼠進(jìn)行Morris水迷宮實驗測試,評價小鼠是否出現(xiàn)學(xué)習(xí)記憶能力損傷,之后取腦組織及海馬組織進(jìn)行HE染色和剛果紅染色,觀察是否出現(xiàn)類似AD的典型病理改變。 BB改善AD學(xué)習(xí)記憶障礙的實驗研究：(1)體外實驗：培養(yǎng)新生大鼠海馬原代神經(jīng)元,設(shè)置正常對照組、Aβ組以及Ap+不同濃度BB組,以BB預(yù)孵育24h后,再以5μmol/L Aβ25-35處理24h模擬AD損傷,MTT法檢測各組神經(jīng)元活力,并確定BB干預(yù)的有效劑量;(2)體內(nèi)試驗：根據(jù)基因型檢測結(jié)果,將小鼠分成陰性對照(CT,溶劑對照)組、模型(AD,溶劑對照)組和模型干預(yù)(AD+BB, BB150mg/kg-bw)組。在小鼠3月齡時,對各組施加相應(yīng)干預(yù),持續(xù)16周,觀察其外觀變化,記錄攝食量,Morris水迷宮試驗評價學(xué)習(xí)記憶能力；之后處死小鼠,稱量小鼠主要臟器重量并計算臟器系數(shù)；對腦皮層及海馬組織進(jìn)行病理檢查,并計數(shù)凋亡、壞死神經(jīng)元數(shù)目。 BB改善AD學(xué)習(xí)記憶相關(guān)機(jī)制的探討：分別從細(xì)胞水平和整體水平進(jìn)行研究。實驗分組及處理同上。采用實時熒光定量PCR (RT-PCR)檢測MAPK/ERK信號通路分子ERK1/2、MEK2,甲基化酶DNMT1、DNMT3a和DNMT3b,組蛋白去乙�；窰DAC1、HDAC2、HDAC3,腦源性神經(jīng)營養(yǎng)因子(BDNF)和泛素羧基末端水解酶(UCH-L1)的mRNA表達(dá)；采用Western blotting檢測ERK1/2、MEK2、DBDNF和UCH-L1蛋白的表達(dá)；檢測小鼠腦組織及血清抗氧化防御系統(tǒng)功能,包括MDA.SOD、還原型GSH和GSH-Px;采用電生理學(xué)方法檢測小鼠海馬CA1區(qū)長時程增強(qiáng)(LTP)。分別從抗氧化、細(xì)胞信號轉(zhuǎn)導(dǎo)、表觀遺傳學(xué)及突觸可塑性的角度探索藍(lán)莓神經(jīng)保護(hù)作用的可能機(jī)制。 結(jié)果 AD轉(zhuǎn)基因小鼠模型的建立：Morris水迷宮檢測結(jié)果顯示,至7月齡時,APP/PS1小鼠定向航行試驗需要耗費較長時間才能到達(dá)平臺,對訓(xùn)練成果記憶穩(wěn)定和牢固程度較差；空間探索試驗結(jié)果顯示,APP/PS1小鼠在目標(biāo)象限區(qū)域的穿越次數(shù)明顯少于正常對照小鼠,出現(xiàn)明顯的學(xué)習(xí)記憶能力損傷。HE染色顯示,7月齡的APP/PS1小鼠腦組織及海馬CA1、CA3區(qū)均出現(xiàn)明顯的神經(jīng)元凋亡、變性,神經(jīng)元減少；剛果紅染色表明,陽性小鼠7月齡時腦組織已出現(xiàn)p淀粉樣蛋白沉積斑塊。 BB改善AD學(xué)習(xí)記憶障礙的實驗研究：(1)細(xì)胞實驗顯示,0.4μg/mlBB可以對抗Ap損傷,提高神經(jīng)元活力,而4μg/ml及以上濃度的BB反而影響細(xì)胞生長,抑制細(xì)胞活力,并且隨濃度的增高,細(xì)胞活力下降程度與BB濃度之間呈現(xiàn)劑量依賴關(guān)系。(2)動物實驗結(jié)果表明,BB干預(yù)可減輕AD模型小鼠老化的部分癥狀和體征,明顯縮短AD小鼠在定向航行試驗中的潛伏時間,增加在空間探索試驗中的目標(biāo)區(qū)域穿越次數(shù),抑制大腦萎縮,病理檢查發(fā)現(xiàn)BB還能減輕AD模型小鼠腦組織神經(jīng)元凋亡、壞死程度。 BB改善AD學(xué)習(xí)記憶相關(guān)機(jī)制的探討：(1)體外實驗：Ap損傷可明顯上調(diào)神經(jīng)元ERK1/2、MEK2及三種HDAC的mRNA和蛋白的表達(dá),同時抑制BDNF、UCH-L1的mRNA及蛋白的表達(dá)；BB干預(yù)后神經(jīng)元ERK1/2、MEK2及三種HDAC表達(dá)上調(diào)的趨勢得到明顯抑制,而BDNF、UCH-L1的mRNA和蛋白表達(dá)明顯增加。(2)動物實驗：與CT組小鼠相比,AD小鼠腦組織中氧化損傷明顯,BB可顯著提高GSH-Px活性,升高還原型GSH,降低MDA水平。RT-PCR和Western blotting檢測結(jié)果與細(xì)胞實驗一致,AD小鼠腦組織ERK1/2、 MEK2和三種HDAC表達(dá)明顯增高,但是BDNF和UCH-L1的表達(dá)明顯減少。BB干預(yù)可有效抑制ERK1/2和MEK2的過度激活,減少HDAC的表達(dá),上調(diào)BDNF和UCH-L1的表達(dá)。電生理學(xué)檢測也顯示,BB可以顯著增強(qiáng)AD小鼠海馬CA1區(qū)LTP。體外及動物實驗均未發(fā)現(xiàn)AD狀態(tài)下以及實施藍(lán)莓干預(yù)對甲基化酶的表達(dá)存在顯著差異。 結(jié)論 (1)該AD轉(zhuǎn)基因小鼠模型繁殖陽性率較穩(wěn)定,AD樣病理及行為特征表現(xiàn)典型,是一種理想的AD動物模型；(2)BB具有拮抗Ap損傷、保護(hù)神經(jīng)元以及改善AD轉(zhuǎn)基因小鼠學(xué)習(xí)記憶能力損傷的作用,但是BB可能存在安全劑量范圍,過高的BB濃度對細(xì)胞生長可能存在不良影響。(3)AD發(fā)病可能通過加劇腦組織的氧化損傷、誘導(dǎo)MAPK/ERK信號通路相關(guān)蛋白的過度表達(dá)和活化、改變某些表觀遺傳學(xué)特征、影響神經(jīng)突觸相關(guān)蛋白的表達(dá),對腦功能造成進(jìn)行性的損傷；而BB可以有效拮抗AD過程中的氧化損傷,調(diào)節(jié)MAPK/ERK信號蛋白的異常表達(dá)和活化,抑制表觀遺傳的異常改變,增強(qiáng)突觸可塑性,從而發(fā)揮其神經(jīng)保護(hù)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.

【學(xué)位授予單位】：天津醫(yī)科大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2013
【分類號】：R285.5;R749.16

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