本文選題：阿爾茨海默病 + Aβ寡聚體�。� 參考：《北京交通大學(xué)》2012年碩士論文

【摘要】：阿爾茨海默病(Alzheimer's disease, AD)是一種與衰老相關(guān)、以認知障礙為主要臨床表現(xiàn)的神經(jīng)退行性疾病。目前公認Ap寡聚體是AD早期神經(jīng)元損傷及病理改變的主要致病物質(zhì)。本研究旨在前期工作基礎(chǔ)上,探討Aβ寡聚體選擇性單抗A8對AD雙轉(zhuǎn)基因模型小鼠的早期治療效果和機制。 選擇4月齡APPswe/PS1△E9雙轉(zhuǎn)基因小鼠,腹腔注射A8單抗。8周后,通過Morris水迷宮實驗(包括定位航行和空間探索實驗)分析轉(zhuǎn)基因小鼠學(xué)習(xí)記憶行為學(xué)的改善情況；以RIPA裂解液制備腦組織勻漿;采用Western blot檢測小鼠腦內(nèi)Aβ42寡聚體、總Aβ、Aβ_(1-42)、Aβ_(1-40)以及磷酸化tau蛋白(p-tau)水平；腦組織超薄切片,在透射電鏡下觀察海馬超微病理改變。利用前期獲得的輕重鏈可變區(qū)基因,構(gòu)建A8的人-鼠嵌合抗體表達載體,dhFr營養(yǎng)缺陷培養(yǎng)基篩選穩(wěn)定的細胞株并初步鑒定。 結(jié)果表明在水迷宮定位航行實驗中,A8組和野生組潛伏期逐漸縮短,在第4d時與對照組相(包括模型組和IgG組)比有統(tǒng)計學(xué)差異(p0.05)。空間探索實驗A8組小鼠潛伏期明顯縮短(p0.05);Western blot結(jié)果表明,A8治療后與對照組相比,在樣品上清與沉淀中檢測到腦內(nèi)Ap寡聚體(p0.01)、總Aβ(p0.01,)、Aβ1-42(p0.05)以及磷酸化(Thr231) tau蛋白水平(p0.01)明顯降低,但是Aβ1-40蛋白水平?jīng)]有明顯變化(p0.05)。透射電鏡結(jié)果表明,A8組小鼠海馬CA1區(qū)內(nèi)突觸數(shù)量高于模型組。間接ELISA檢測人-鼠嵌合抗體Ch-A8特異性識別Aβ1-42寡聚體(A450=2.2265±0.3353),與鼠源A8(A450=2.525±02153)相近。 綜上所述,A8單克隆抗體早期外周給藥對AD雙轉(zhuǎn)基因小鼠有明顯的治療效果,前期實驗獲得的A8輕重鏈可變區(qū)基因能夠以嵌合抗體形式正確表達。為進一步人源化改造及AD治療藥物的開發(fā)提供了有效依據(jù)。
[Abstract]:Alzheimer's disease (ADD) is a neurodegenerative disease associated with aging and characterized by cognitive impairment. At present, it is widely accepted that AP oligomer is the main pathogenicity of neuronal injury and pathological changes in early AD. The aim of this study was to investigate the early therapeutic effect and mechanism of A 尾 oligomer selective monoclonal antibody A8 on AD double transgenic mice. 4-month-old APPswe/PS1 E9 transgenic mice were selected. After intraperitoneal injection of A8 monoclonal antibody, the improvement of learning and memory behavior of transgenic mice was analyzed by Morris water maze test (including positioning navigation and space exploration experiment). Brain tissue homogenate was prepared from RIPA cleavage fluid, A 尾 42 oligomer, total A 尾 1 42 A 尾 1 40) and phosphorylated tau protein p-tau. were detected by Western blot, and ultrathin sections of brain tissue were observed under transmission electron microscope. Using the variable region gene of light and light chain obtained in the early stage, we constructed the human mouse chimeric antibody expression vector of A8 and screened the stable cell line on the medium of nutrition deficiency of DHFR and identified it preliminarily. The results showed that the latency of A8 group and wild group was shortened gradually in the water maze navigation experiment, and there was a significant difference between the two groups on the 4th day (including model group and IgG group). Compared with the control group, the incubation period of A8 group was significantly shorter than that of the control group. Compared with the control group, the levels of Ap oligomer p0.01, total A 尾 -p0.01A 尾 1-42P 0.05 and phosphorylated Thr231) tau protein in the brain were significantly decreased in the spatial exploration experiment A8 group compared with the control group, and the levels of phosphorylated Thr231) tau protein were significantly decreased in the A8 group compared with those in the control group. However, the level of A 尾 1-40 protein did not change significantly (p 0.05). The results of transmission electron microscope showed that the number of synapses in the hippocampal CA1 region of A8 group was higher than that in the model group. Indirect ELISA was used to detect the specific recognition of A 尾 1-42 oligodeoxynucleotides (A 尾 1-42 oligodeoxynucleotides) by Ch-A8, which was similar to that of murine A8(A450=2.525 鹵0.2153 (2.2265 鹵0.3353). To sum up, early peripheral administration of monoclonal antibody to A8 has obvious therapeutic effect on AD double transgenic mice, and the variable region gene of A8 light and heavy chain obtained in previous experiments can be correctly expressed in the form of chimeric antibody. It provides an effective basis for further humanization and the development of AD treatment drugs.
【學(xué)位授予單位】：北京交通大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2012
【分類號】：R749.16

【參考文獻】

相關(guān)碩士學(xué)位論文 前1條

1 王鑫;Aβ寡聚體單克隆抗體的制備及功能研究[D];北京交通大學(xué);2009年

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本文編號：1872363