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PTK2B通过LRP-1对Aβ诱导的认知功能障碍小鼠血液和脑组织中Aβ水平及其行为的影响

郝凯敏, 刘镇, 王浩玉, 李坤, 祁文秀^*

山西医科大学汾阳学院，汾阳 032200

摘要

本文旨在探讨ptk2b基因及其表达产物PTK2B (protein tyrosine kinase 2 beta)蛋白与低密度脂蛋白受体相关蛋白1 (low density lipoprotein receptor-related protein-1, LRP-1)的关系，分析认知功能障碍小鼠血液和脑组织中的Aβ变化与ptk2b基因的关系。将64只三月龄C57BL/6J小鼠随机分成实验组和对照组，所有动物均行侧脑室插管术。实验组小鼠均经侧脑室(i.c.v.)注射凝聚态Aβ1-42 (0.1 μg/μL, 3.6 μL)建立认知功能障碍模型，3 d后，分别注射PTK2B抑制剂PF431396 (15 μg/mL，Aβ + PF组)、PTK2B激动剂PMA (18.75 μg/mL，Aβ + PMA组)、LRP-1抑制剂RAP (0.2 μg/mL，Aβ + RAP组)以及生理盐水(Aβ + NS组)；对照组则经侧脑室插管注射NS，3 d后分别i.c.v.注射等量的PF431396 (PF组)、PMA (PMA组)、RAP (RAP组)以及NS (NS组)。每组均注射2 μL。注射1周后用Morris水迷宫对小鼠进行认知行为学功能检测，用免疫组织化学、ELISA以及Western blot检测血液和海马组织中Aβ1-42、LRP-1、PTK2B的表达水平，用qRT-PCR检测海马ptk2b mRNA的表达。结果显示，Aβ诱导的认知功能障碍模型小鼠海马组织中Aβ1-42水平升高，血液中则降低，同时海马中LRP-1蛋白表达水平下调，海马PTK2B和ptk2b mRNA的表达水平则上调；PTK2B抑制剂可下调认知功能障碍小鼠海马的PTK2B以及ptk2b mRNA的表达，上调海马LRP-1表达，降低海马组织中Aβ1-42水平，而升高血液中Aβ1-42水平，同时改善小鼠的认知功能；PTK2B激动剂的作用则相反。用LRP-1抑制剂干预下调海马中的LRP-1水平后，海马Aβ1-42水平升高而血液中Aβ1-42水平则降低，同时小鼠的认知行为学功能则明显降低，而海马中PTK2B以及ptk2b mRNA表达却没有明显变化。以上结果提示，PTK2B可能通过下调认知功能障碍模型小鼠脑组织中的LRP-1而升高海马组织Aβ1-42水平、降低血液中的Aβ1-42水平，进而影响其认知功能。

关键词： 阿尔茨海默病; 淀粉样β蛋白; 低密度脂蛋白受体相关蛋白1; PTK2B; C57BL/6J小鼠

分类号：R742

PTK2B affects the levels of Aβ in blood and brain and behavioral functions via targeting LRP-1 transporter in Aβ-induced cognitive dysfunction mice

HAO Kai-Min, LIU Zhen, WANG Hao-Yu, LI Kun, QI Wen-Xiu^*

Fenyang College of Shanxi Medical University, Fenyang 032200, China

Abstract

The aim of the present study was to explore the correlation between ptk2b/PTK2B (protein tyrosine kinase 2 beta, a ptk2b-encoded protein) and the level of low density lipoprotein receptor-related protein-1 (LRP-1), as well as to uncover the relationship between the changes in beta amyloid protein (Aβ) levels in blood and brain and the expression of ptk2b in Aβ-induced cognitive dysfunction mice. A total of 64 3-month-old C57BL/6J mice were divided randomly into the experimental group and control group. All mice underwent the intracerebroventricular (i.c.v.) intubation. Mice in the experimental group received the i.c.v. infusion of oligomeric Aβ1-42 (0.1 μg/μL, 3.6 μL) to construct the cognitively impaired models, and three days later, those mice were further injected with PF431396 (an inhibitor of PTK2B, 15 μg/mL, Aβ + PF group), phorbol-12-myristate-13-acetate (PMA, an agonist of PTK2B, 18.75 μg/mL, Aβ + PMA group), RAP (an inhibitor of LRP-1, 0.2 μg/mL, Aβ + RAP group) or normal saline (Aβ + NS group). For mice in the control group, they underwent the i.c.v. infusion of NS, and 3 days later, they were additionally injected with PF431396 (PF group), PMA (PMA group), RAP (RAP group) or NS (NS group) in the volume of 2 μL. One week later, all mice were subjected to the determination of behavioral function in Morris water maze and the measurement of expression of Aβ1-42, LRP-1 and PTK2B in blood and hippocampus using immunohistochemistry (IHC) staining, enzyme-linked immunosorbent assay (ELISA) and Western blot, and the measurement of mRNA expression of ptk2b in hippocampus using qRT-PCR. The results showed that the infusion of Aβ induced an increase of Aβ1-42 level in hippocampus and a decrease in blood, with the down-regulation of LRP-1 protein expression in hippocampus and up-regulation of mRNA and protein expression of ptk2b in hippocampus. For cognitively impaired mice, intervention of PF431396 caused the down-regulation of protein and mRNA expression of ptk2b in the hippocampus, while LRP-1 in hippocampus was up-regulated with a decrease in the level of Aβ1-42 in hippocampus and an increase in the level of Aβ1-42 in the blood, as well as significant improvement in cognitive function, while the administration of PMA resulted in the opposite changes. Moreover, the administration of RAP triggered the down-regulation of LRP-1 expression in hippocampus and an increase in the level of Aβ1-42 in hippocampus and a decrease in the level of Aβ1-42 in blood, with the deterioration of the behavioral functions, while protein and mRNA expression of ptk2b in hippocampus showed no evident changes. These results suggest that, in cognitively impaired mice, PTK2B, possibly via down-regulating LRP-1, increases the Aβ1-42 level in brain, but decreases the Aβ1-42 level in blood, thereby deteriorating the cognitive and behavioral functions of mice.

Key words: Alzheimer’s disease; beta amyloid protein; low density lipoprotein receptor-related protein-1; PTK2B; C57BL/6J mice

收稿日期：2020-10-09　　录用日期：2021-01-25

通讯作者：祁文秀　　E-mail: fycqwx@163.com

DOI: 10.13294/j.aps.2021.0069

引用本文：

郝凯敏, 刘镇, 王浩玉, 李坤, 祁文秀. PTK2B通过LRP-1对Aβ诱导的认知功能障碍小鼠血液和脑组织中Aβ水平及其行为的影响[J]. 生理学报 2021; 73 (5): 845-854.

HAO Kai-Min, LIU Zhen, WANG Hao-Yu, LI Kun, QI Wen-Xiu. PTK2B affects the levels of Aβ in blood and brain and behavioral functions via targeting LRP-1 transporter in Aβ-induced cognitive dysfunction mice. Acta Physiol Sin 2021; 73 (5): 845-854 (in Chinese with English abstract).