Ogata Seiryo, Ito Shingo, Masuda Takeshi, Ohtsuki Sumio
Department of Pharmaceutical Microbiology, Graduate School of Pharmaceutical Sciences, Kumamoto University, 5-1 Oe-honmachi, Chuo-ku, Kumamoto, 862-0973, Japan.
Department of Environmental Medicine and Molecular Toxicology, Tohoku University Graduate School of Medicine, 2-1 Seiryo-machi, Aoba-ku, Sendai, 980-8575, Japan.
Fluids Barriers CNS. 2024 Dec 19;21(1):106. doi: 10.1186/s12987-024-00609-6.
Protein expression analysis of isolated brain microvessels provides valuable insights into the function of the blood-brain barrier (BBB). However, isolation of brain microvessels from human brain tissue, particularly in small quantities, poses significant challenges. This study presents a method for isolating brain microvessels from a small amount of frozen human brain tissue, adapting techniques from an established mouse brain capillary isolation method.
Brain microvessel fractions were obtained from approximately 0.3 g of frozen human brain tissue (frontal cortex) using a bead homogenizer for homogenization, followed by purification with a combination of cell strainers and glass beads. Protein expression in the isolated human microvessel fractions and whole-brain lysates was analyzed by western blot and proteomic analysis.
Microscopic imaging confirmed the successful isolation of brain microvessels from frozen human brain tissue. Protein quantification assays demonstrated that the microvessel fraction yielded sufficient protein for detailed expression analysis. Western blot analysis revealed an enrichment of BBB-selective proteins including multidrug resistance 1 (MDR1)/ATP-binding cassette sub-family B member 1 (ABCB1), glucose transporter protein type 1 (GLUT1)/solute carrier family 2 member 1 (SLC2A1), and claudin 5 (CLDN5), in the brain microvessel fraction compared to whole-brain lysates. Multiple reaction monitoring quantification of six BBB-selective proteins-MDR1, breast cancer resistance protein (BCRP)/ATP binding cassette subfamily G member 2 (ABCG2), GLUT1, monocarboxylate transporter 1 (MCT1)/solute carrier family 16 member 1 (SLC16A1), transferrin receptor, and CLDN5-revealed expression levels consistent with those observed in larger human brain samples. Sequential Window Acquisition of all Theoretical Mass Spectra (SWATH-MS)-based quantitative proteomics further demonstrated significant enrichment of human microvascular endothelial cells in the isolated fraction, corroborating the findings from mouse models.
We successfully developed a method for isolation of brain microvessels from a small amount of frozen human brain tissue, facilitating detailed study of BBB proteome in aging or pathological conditions. This technique provides valuable insights into BBB dysfunction in central nervous system disorders and holds potential for improving brain-targeted drug delivery strategies.
对分离出的脑微血管进行蛋白质表达分析，可为血脑屏障（BBB）的功能提供有价值的见解。然而，从人脑组织中分离脑微血管，尤其是少量组织，面临着重大挑战。本研究提出了一种从少量冷冻人脑组织中分离脑微血管的方法，该方法借鉴了已有的小鼠脑毛细血管分离方法。
使用珠磨匀浆机对约0.3g冷冻人脑组织（额叶皮质）进行匀浆，获得脑微血管级分，随后通过细胞筛网和玻璃珠相结合的方式进行纯化。通过蛋白质印迹法和蛋白质组学分析，对分离出的人脑微血管级分和全脑裂解物中的蛋白质表达进行分析。
显微镜成像证实成功从冷冻人脑组织中分离出脑微血管。蛋白质定量分析表明，微血管级分产生了足够的蛋白质用于详细的表达分析。蛋白质印迹分析显示，与全脑裂解物相比，脑微血管级分中血脑屏障选择性蛋白富集，包括多药耐药蛋白1（MDR1）/ATP结合盒亚家族B成员1（ABCB1）、葡萄糖转运蛋白1型（GLUT1）/溶质载体家族2成员1（SLC2A1）和紧密连接蛋白5（CLDN5）。对六种血脑屏障选择性蛋白——MDR1、乳腺癌耐药蛋白（BCRP）/ATP结合盒亚家族G成员2（ABCG2）、GLUT1、单羧酸转运蛋白1（MCT1）/溶质载体家族16成员1（SLC16A1）、转铁蛋白受体和CLDN5进行多反应监测定量分析，结果显示其表达水平与在较大人脑样本中观察到的一致。基于所有理论质谱的顺序窗口采集（SWATH-MS）定量蛋白质组学进一步证明，分离级分中人类微血管内皮细胞显著富集，这与小鼠模型的研究结果一致。
我们成功开发了一种从少量冷冻人脑组织中分离脑微血管的方法，有助于在衰老或病理条件下对血脑屏障蛋白质组进行详细研究。该技术为深入了解中枢神经系统疾病中的血脑屏障功能障碍提供了有价值的见解，并有望改善脑靶向给药策略。
