ADAM10基因缺失对成年小鼠大脑Aβ肽的产生和认知功能的影响

发布时间：2018-08-11 14:26

【摘要】：目的：淀粉样前体蛋白（APP）经过β-分泌酶和γ-分泌酶水解，释放出Aβ肽。Aβ肽是淀粉斑的核心成分，它的过多生成或聚集被认为是阿尔茨海默病致病的主要原因。APP蛋白还有另一条非淀粉样蛋白加工途径，在这条途径中，它被α-分泌酶和γ-分泌酶连续水解。由于α-分泌酶的水解APP位点在Aβ肽内部，因此，可以减少Aβ肽的生成。目前，已经证实去整合素和金属蛋白酶10（ADAM10）是神经元的组成型APP α-分泌酶。但是，ADAM10缺失究竟对成年神经细胞的功能造成何种影响迄今不清楚，为此，我们利用本室建立的成年大脑神经细胞特异性ADAM10基因敲除小鼠（ADAM10cKO）模型研究ADAM10基因缺失对APP代谢水平和小鼠学习记忆的影响。方法：利用PCR方法鉴定ADAM10cKO小鼠的基因型。鉴定出的纯合子小鼠用于后续的实验研究。按照常规的组织学方法，取3、6、9和12月龄ADAM10cKO小鼠和野生型C57BL/6J小鼠（WT）的大脑，经过固定、脱水、透明、石蜡包埋、切片和HE染色，观察ADAM10cKO小鼠和WT小鼠大脑海马和皮层组织的形态结构，以及皮层第5层和海马神经元数量的变化。分别取7、9、11、12、13和15月龄ADAM10cKO小鼠和ADAM10Fl/Fl对照小鼠的大脑，经过固定、脱水、透明、石蜡包埋、切片和免疫组织化学染色，观察β淀粉样蛋白（Aβ）在大脑皮层和海马组织中的分布，并分析Aβ沉积与年龄的关系。分别提取20月龄的ADAM10cKO小鼠和ADAM10Fl/Fl小鼠的皮层和海马组织蛋白，采用Western blotting方法定量测定APP和Aβ肽的含量，分析ADAM10基因缺失对APP代谢的影响。分别取10月龄ADAM10cKO小鼠和WT小鼠各9只进行水迷宫实验，通过检测逃避潜伏期、目标象限（原先放置平台的象限）停留的时间百分比和1min内穿过平台次数评价两组小鼠的学习和记忆能力。结果：HE染色结果表明，3、6、9和12月龄ADAM10cKO小鼠大脑皮层和海马的形态结构未见明显异常，，但是，皮层第Ⅴ层的神经元数量随着年龄的增大而减少，海马神经细胞减少更为明显。免疫组织化学实验结果显示，7、9、11、13和15月龄ADAM10cKO小鼠与同龄的ADAM10Fl/Fl对照小鼠相比，其大脑皮层和海马未见淀粉斑，但在神经细胞内可溶性Aβ肽呈年龄依赖性增加。可溶性Aβ肽的含量在两种小鼠的海马和皮层的神经细胞中表现出差异。Western blotting实验结果表明，ADAM10cKO小鼠与ADAM10FL/FL小鼠比较，其成熟与非成熟的全长APP蛋白含量在海马中分别减少73.0%和62.3%（P㩳0.01），但是，在大脑皮层中，成熟与非成熟的全长APP蛋白含量变化不明显；ADAM10cKO小鼠大脑皮层和海马中Aβ肽总含量分别增加20.45%和27.21%（P0.05和P0.01），皮层中Aβ1-38、 A β1-40和A β1-42肽的含量分别增加32.85%、25.37%和80.95%(P0.05，P0.05和P0.01)，海马中Aβ1-38、Aβ1-40和Aβ1-42肽的含量分别增加41.67%、38.36%和22.22%（P㩳0.05），呈现明显差异。水迷宫实验结果显示,在训练期间，ADAM10cKO小鼠的平均潜伏期比WT小鼠的时间长，第2天和第3天分别表现出明显差异（P㩳0.05和P㩳0.01）。在测试期间，ADAM10cKO小鼠和WT小鼠相比，其平均穿越平台的次数少，并且，平均停留目标象限时间的百分比低，均呈现明显差异（P㩳0.05）。结论：ADAM10基因缺失导致成年小鼠大脑皮层和海马的神经细胞数目明显减少。APP含量在大脑皮层中无变化，而在海马中明显减少，可能是海马神经元过多丢失所致。可溶性Aβ肽含量呈年龄依赖性增加，是神经退化和认知功能受到损害的主要原因。
[Abstract]:OBJECTIVE: Amyloid precursor protein (APP) is hydrolyzed by beta-secretase and gamma-secretase to release a beta peptide. A beta peptide is the core component of amyloid plaque. Its excessive production or aggregation is considered to be the main cause of Alzheimer's disease. APP protein also has another non-amyloid protein processing pathway, in which it is processed by alpha-secretase and gamma-secretase. Continuous hydrolysis of gamma-secretase. As the APP site of alpha-secretase is located within the A-beta peptide, the production of A-beta peptide can be reduced. At present, it has been confirmed that disintegrin and metalloproteinase 10 (ADAM10) are constitutive APP-secretases of neurons. However, it is unclear how the deletion of ADAM10 affects the function of adult neurons. In order to investigate the effects of ADAM10 gene deletion on APP metabolism and learning and memory in adult brain neuron-specific ADAM10 knockout mice (ADAM10cKO), we established a model in our laboratory.
METHODS: The genotypes of ADAM10cKO mice were identified by PCR. The homozygous mice were used for follow-up study. According to conventional histological methods, the brains of ADAM10cKO mice and wild type C57BL/6J mice (WT) were taken and fixed, dehydrated, transparent, paraffin embedded, sliced and stained with HE. The brain of ADAM10cKO mice and A DAM10Fl/Fl control mice aged 7,9,11,12,13 and 15 months were fixed, dehydrated, transparent, paraffin embedded, sectioned and immunohistochemical stained to observe the changes of amyloid beta (Abeta) in the brain. The protein of cortex and hippocampus of ADAM10 cKO mice and ADAM10 Fl/Fl mice aged 20 months were extracted respectively. The content of APP and A beta peptide was quantitatively determined by Western blotting. The effect of ADAM10 gene deletion on APP metabolism was analyzed. The learning and memory abilities of the two groups were evaluated by detecting the escape latency, the percentage of time spent in the target quadrant (the quadrant where the platform was originally placed) and the number of times crossed the platform in one minute.
Results: The results of HE staining showed that the morphological structure of cerebral cortex and hippocampus of ADAM10cKO mice aged 3,6,9 and 12 months had no obvious abnormality, but the number of neurons in the fifth cortex decreased with the increase of age, and the decrease of neurons in hippocampus was more obvious. Compared with A DAM10Fl/Fl control mice of the same age, there was no amyloid plaque in cerebral cortex and hippocampus, but soluble A-beta peptide in neurons increased in an age-dependent manner. Compared with mice, the content of mature and immature full-length APP protein in hippocampus decreased by 73.0% and 62.3% (P? 0.01), but the content of mature and immature full-length APP protein did not change significantly in cerebral cortex; the total content of Abeta peptide in cerebral cortex and hippocampus of ADAM10cKO mice increased by 20.45% and 27.21% (P 0.05 and P 0.01), respectively, and Abeta 1-3 in cortex. The contents of 8, A-beta-1-40 and A-beta-42 peptides increased by 32.85%, 25.37% and 80.95% (P 0.05, P 0.05 and P 0.01), respectively. The contents of A-beta-38, A-beta-40 and A-beta-42 peptides in hippocampus increased by 41.67%, 38.36% and 22.22% (P?0.05), respectively. The results of water maze experiment showed that the average latency of ADAM-10 cKO mice was longer than that of WT mice. During the test period, ADAM 10cKO mice and WT mice crossed the platform fewer times on average, and the percentage of average dwell time in the target quadrant was lower (P?0.05).
CONCLUSION: The deletion of ADAM10 gene results in a significant decrease in the number of neurons in the cerebral cortex and hippocampus of adult mice. APP content has no change in the cerebral cortex, but decreased significantly in the hippocampus, possibly due to excessive loss of neurons in the hippocampus. Main cause.
【学位授予单位】：福建医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R749.16

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