Ogata Seiryo, Ito Shingo, Masuda Takeshi, Ohtsuki Sumio
Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, Kumamoto, Japan.
Department of Pharmaceutical Microbiology, Faculty of Life Sciences, Kumamoto University, Kumamoto, Japan.
J Cereb Blood Flow Metab. 2021 May;41(5):1026-1038. doi: 10.1177/0271678X20941449. Epub 2020 Jul 23.
Isolated brain capillaries are essential for analyzing the changes of protein expressions at the blood-brain barrier (BBB) under pathological conditions. The standard brain capillary isolation methods require the use of at least five mouse brains in order to obtain a sufficient amount and purity of brain capillaries. The purpose of this study was to establish a brain capillary isolation method from a single mouse brain for protein expression analysis. We successfully isolated brain capillaries from a single frozen mouse brain by using a bead homogenizer in the brain homogenization step and combination of cell strainers and glass beads in the purification step. Western blot and proteomic analysis showed that proteins expressed at the BBB in mouse brain capillaries isolated by the developed method were more enriched than those isolated from a pool of five mouse brains by the standard method. By using the developed method, we further verified the changes in expression of BBB proteins in -deficient mouse. The developed method is useful for the analysis of various mice models with low numbers and enables us to understand, in more detail, the physiology and pathology of BBB.
分离的脑毛细血管对于分析病理条件下血脑屏障（BBB）处蛋白质表达的变化至关重要。标准的脑毛细血管分离方法需要使用至少五只小鼠的大脑，以获得足够数量和纯度的脑毛细血管。本研究的目的是建立一种从单个小鼠大脑中分离脑毛细血管用于蛋白质表达分析的方法。我们通过在脑匀浆步骤中使用珠子匀浆器以及在纯化步骤中结合细胞滤网和玻璃珠，成功地从单个冷冻小鼠大脑中分离出了脑毛细血管。蛋白质印迹和蛋白质组学分析表明，通过所开发的方法分离的小鼠脑毛细血管中BBB处表达的蛋白质比通过标准方法从五只小鼠大脑的混合样本中分离的蛋白质更丰富。通过使用所开发的方法，我们进一步验证了β-缺陷小鼠中BBB蛋白质表达的变化。所开发的方法对于分析数量较少的各种小鼠模型很有用，并且使我们能够更详细地了解BBB的生理学和病理学。
