Aβ寡聚体选择性单抗对阿尔茨海默病双转基因小鼠治疗效果的研究

发布时间：2018-05-11 04:15

本文选题：阿尔茨海默病 + Aβ寡聚体　；参考：《北京交通大学》2012年硕士论文

【摘要】：阿尔茨海默病(Alzheimer's disease, AD)是一种与衰老相关、以认知障碍为主要临床表现的神经退行性疾病。目前公认Ap寡聚体是AD早期神经元损伤及病理改变的主要致病物质。本研究旨在前期工作基础上,探讨Aβ寡聚体选择性单抗A8对AD双转基因模型小鼠的早期治疗效果和机制。选择4月龄APPswe/PS1△E9双转基因小鼠,腹腔注射A8单抗。8周后,通过Morris水迷宫实验(包括定位航行和空间探索实验)分析转基因小鼠学习记忆行为学的改善情况；以RIPA裂解液制备脑组织匀浆;采用Western blot检测小鼠脑内Aβ42寡聚体、总Aβ、Aβ_(1-42)、Aβ_(1-40)以及磷酸化tau蛋白(p-tau)水平；脑组织超薄切片,在透射电镜下观察海马超微病理改变。利用前期获得的轻重链可变区基因,构建A8的人-鼠嵌合抗体表达载体,dhFr营养缺陷培养基筛选稳定的细胞株并初步鉴定。结果表明在水迷宫定位航行实验中,A8组和野生组潜伏期逐渐缩短,在第4d时与对照组相(包括模型组和IgG组)比有统计学差异(p0.05)。空间探索实验A8组小鼠潜伏期明显缩短(p0.05);Western blot结果表明,A8治疗后与对照组相比,在样品上清与沉淀中检测到脑内Ap寡聚体(p0.01)、总Aβ(p0.01,)、Aβ1-42(p0.05)以及磷酸化(Thr231) tau蛋白水平(p0.01)明显降低,但是Aβ1-40蛋白水平没有明显变化(p0.05)。透射电镜结果表明,A8组小鼠海马CA1区内突触数量高于模型组。间接ELISA检测人-鼠嵌合抗体Ch-A8特异性识别Aβ1-42寡聚体(A450=2.2265±0.3353),与鼠源A8(A450=2.525±02153)相近。综上所述,A8单克隆抗体早期外周给药对AD双转基因小鼠有明显的治疗效果,前期实验获得的A8轻重链可变区基因能够以嵌合抗体形式正确表达。为进一步人源化改造及AD治疗药物的开发提供了有效依据。
[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 卤0.2153 (2.2265 卤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.
【学位授予单位】：北京交通大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R749.16

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