【摘要】：【目的】β淀粉樣蛋白（β-amyloid，Aβ）代謝異常是阿爾茨海默�。ˋlzheimer’sdisease，，AD）發(fā)病機(jī)制的核心環(huán)節(jié)。Aβ的生成和清除失平衡導(dǎo)致其在腦內(nèi)異常增多并聚集，觸發(fā)了一系列病理生理級(jí)聯(lián)反應(yīng)，包括突觸功能障礙、膠質(zhì)細(xì)胞活化誘發(fā)的炎癥反應(yīng)等，最終導(dǎo)致神經(jīng)元損傷并引起癡呆。本研究以5XFAD轉(zhuǎn)基因小鼠為動(dòng)物模型，觀察雷公藤氯內(nèi)酯醇（tripchlorolide，T_4）對(duì)該轉(zhuǎn)基因小鼠認(rèn)知功能的影響，并分別從突觸可塑性、Aβ代謝和神經(jīng)炎癥的角度較為深入的探討T_4作用的可能分子機(jī)制。 【方法】SPF級(jí)5月齡雄性5XFAD轉(zhuǎn)基因小鼠和野生型小鼠，各自分別隨機(jī)分為溶媒對(duì)照組（0.9%NaCl）、T_4處理組（5μg/kg，25μg/kg），每組8～11只，腹腔注射給藥，隔日1次，共60天。 1、采用Y迷宮和Morris水迷宮兩種行為學(xué)實(shí)驗(yàn)，檢測(cè)動(dòng)物學(xué)習(xí)記憶功能。 2、電鏡下觀察海馬CA1區(qū)突觸超微結(jié)構(gòu)；Western blot法檢測(cè)海馬和皮層突觸可塑性相關(guān)蛋白synaptophsin、 PSD-95、 p-NMDAR1/NMDAR1、p-CaMKIIα/CaMKIIα、p-CREB/CREB和PI3K/Akt/mTOR信號(hào)通路中pAkt/Akt、pmTOR/mTOR的表達(dá)。 3、通過免疫組化、ELISA和Western blot檢測(cè)腦內(nèi)Aβ及其生成和降解的關(guān)鍵酶（包括β分泌酶、α分泌酶、NEP和IDE）的表達(dá)。 4、通過免疫組化觀察腦內(nèi)小膠質(zhì)細(xì)胞和星形膠質(zhì)細(xì)胞的活化情況，通過Western blot檢測(cè)腦內(nèi)GFAP的表達(dá)，利用real-time PCR檢測(cè)海馬炎癥介質(zhì)的mRNA水平。 【結(jié)果】 1、T_4可改善5XFAD轉(zhuǎn)基因小鼠視空間學(xué)習(xí)記憶障礙。 2、T_4可改善5XFAD轉(zhuǎn)基因小鼠海馬突觸超微結(jié)構(gòu)異常，增加腦內(nèi)突觸可塑性相關(guān)蛋白的表達(dá)，同時(shí)增加pAkt/Akt、pmTOR/mTOR的表達(dá)。 3、T_4能夠減少5XFAD轉(zhuǎn)基因小鼠腦內(nèi)Aβ沉積，降低BACE1及其酶切產(chǎn)物sAPP-β、CTF-β的表達(dá)，增加NEP的表達(dá)，但對(duì)APP、CTF-α、ADAM10、ADAM17、IDE的表達(dá)無影響。 4、T_4能夠抑制5XFAD轉(zhuǎn)基因小鼠腦內(nèi)小膠質(zhì)細(xì)胞活化，下調(diào)炎癥介質(zhì)IL-1β、TNF-α、IL-6、TGF-β、CCL2、NOS2的mRNA水平，但對(duì)星形膠質(zhì)細(xì)胞活化無明顯影響。 【結(jié)論】5XFAD轉(zhuǎn)基因小鼠存在視空間學(xué)習(xí)記憶障礙、突觸功能損害、Aβ沉積和神經(jīng)炎癥反應(yīng)。T_4可能通過活化PI3K-Akt-mTOR通路促進(jìn)突觸可塑性相關(guān)蛋白的合成；抑制BACE1活性，增加NEP表達(dá)，減少Aβ生成并促進(jìn)其降解清除；抑制小膠質(zhì)細(xì)胞活化所致的神經(jīng)炎癥反應(yīng)，從而改善該轉(zhuǎn)基因小鼠的認(rèn)知功能障礙。因此，T_4有望成為AD治療有前途的候選藥物。
[Abstract]:[objective] abnormal metabolism of 尾 -amyloid protein (尾 -amyloid 尾) is the key link in the pathogenesis of Alzheimer's disease (Alzheimer'sdisease,AD). It triggers a series of pathophysiological cascade reactions, including synaptic dysfunction and inflammation induced by the activation of glial cells, leading to neuronal damage and dementia. The effects of tripterygium wilfordii chloride lactone (tripchlorolide,T_4) on the cognitive function of 5XFAD transgenic mice were studied, and the synaptic plasticity was observed. The possible molecular mechanism of T4 was discussed in detail from the perspective of A 尾 metabolism and neuroinflammation. [methods] SPF grade 5-month-old male 5XFAD transgenic mice and wild-type mice were randomly divided into two groups: solvent control group (0.9%NaCl), T4 treated group (5 渭 g / kg ~ 25 渭 g/kg), each group (n = 8). Once every other day for 60 days. 1. Two behavioral experiments, Y maze and Morris water maze, were used to detect the learning and memory function of animals. 2. Ultrastructure of synapses in hippocampal CA1 area was observed under electron microscope. The expression of pAkt/Akt,pmTOR/mTOR in hippocampal and cortical synaptic plasticity associated protein (synaptophsin, PSD-95, p-NMDAR1 / p-CaMKII 偽 / CaMKII 偽, p-CREB/CREB and PI3K/Akt/mTOR signaling pathway) was detected by Western blot method. 3The expression of A 尾 and its key enzymes (including 尾 -secretase, 偽 -secretase, NEP and IDE) in brain were detected by immunohistochemistry, ELISA and Western blot. (4) the activation of microglia and astrocytes in brain was observed by immunohistochemistry, the expression of GFAP in brain was detected by Western blot, and the mRNA level of inflammatory mediators in hippocampus was detected by real-time PCR. [results] 1. Tip4 could improve the visual spatial learning and memory impairment of 5XFAD transgenic mice. 2TX _ 4 could improve the synaptic ultrastructure abnormality, increase the expression of synaptic plasticity related protein and increase the expression of pAkt/Akt,pmTOR/mTOR in hippocampal synaptic ultrastructure of 5XFAD transgenic mice. 3T _ 4 could reduce A 尾 deposition in brain of 5XFAD transgenic mice, decrease the expression of sAPP- 尾 and CTF- 尾, and increase the expression of NEP, but had no effect on the expression of APP,CTF- 偽 and ADAM10,ADAM17,IDE. 4T _ 4 could inhibit the activation of microglia in the brain of 5XFAD transgenic mice and down-regulate the mRNA levels of inflammatory mediators IL-1 尾, TNF- 偽, IL-6,TGF- 尾 and CCL2,NOS2, but had no effect on the activation of astrocytes. [conclusion] 5XFAD transgenic mice have visual spatial learning and memory impairment, synaptic dysfunction, A 尾 deposition and neuroinflammatory response. Tap4 may promote the synthesis of synaptic plasticity related proteins by activating PI3K-Akt-mTOR pathway. Inhibition of BACE1 activity, increase of NEP expression, reduction of A 尾 production and promotion of its degradation and clearance, inhibition of neuroinflammation induced by activation of microglia, and improvement of cognitive impairment in transgenic mice. Therefore, T-d-4 is expected to be a promising drug candidate for AD therapy.
【學(xué)位授予單位】：福建醫(yī)科大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2013
【分類號(hào)】：R749.16

【參考文獻(xiàn)】

相關(guān)期刊論文 前2條

1 胡小令,呂誠(chéng),萬衛(wèi),張憲郁;雷公藤多甙對(duì)Alzheimer病大鼠學(xué)習(xí)記憶和小膠質(zhì)細(xì)胞的影響[J];中國(guó)老年學(xué)雜志;2005年02期

2 呂誠(chéng);胡小令;萬斌;楊寶林;;雷公藤內(nèi)酯醇對(duì)阿爾茨海默病模型大鼠學(xué)習(xí)記憶和海馬核因子-κB表達(dá)的影響[J];中國(guó)老年學(xué)雜志;2009年17期

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