小鼠脑血管平滑肌细胞和内皮细胞的联合提取与鉴定  

Combined extraction and identification of mouse brain vascular smooth muscle cells and endothelial cells

作　　者：姚智超 杨阳 霍丽蓉[1] YAO Zhichao;YANG Yang;HUO Lirong(Central Laboratory,Fuxing Hospital,Capital Medical University,Beijing 100038,China)

机构地区：[1]首都医科大学附属复兴医院中心实验室,北京市100038

出　　处：《中国动脉硬化杂志》2024年第9期790-797,共8页Chinese Journal of Arteriosclerosis

基　　金：北京市自然科学基金面上项目(7182065)。

摘　　要：[目的]建立一种高效且稳定的原代小鼠脑血管平滑肌细胞和内皮细胞的分离方法,为脑血管病发病机制和治疗方法的探究提供实验材料。[方法]通过葡聚糖梯度离心联合酶消化法分离脑血管平滑肌细胞,再通过免疫磁珠分选分离脑血管内皮细胞。使用倒置相差显微镜观察两种细胞的形态和生长特征,通过免疫荧光鉴定纯度,通过CCK-8法观察传代后生长和增殖特性。在细胞功能层面,通过血管形成实验评估内皮细胞血管形成能力,通过迁移实验评估平滑肌细胞对血小板源性生长因子(PDGF)的反应性。[结果]采用本方法分离出的两种细胞生长旺盛,状态良好。平滑肌细胞表现出典型的“峰谷状”生长,免疫荧光结果显示胞质内平滑肌特异性α-SMA和SM22α表达阳性;内皮细胞表现出典型的“铺路石”样生长,血小板内皮细胞黏附分子CD31和闭锁小带蛋白1表达阳性。[结论]本研究建立了一种高效同时分离两种脑血管细胞的可靠方法,分离细胞纯度高,活性良好,且传代后特征稳定,足以满足后续实验需求。Aim To establish an efficient and stable isolation method of primary mouse brain vascular smooth muscle cells and endothelial cells,and provide experimental materials for the investigation of pathogenesis and treatment of brain vascular diseases.Methods Brain vascular smooth muscle cells were isolated by dextran gradient centrifugation with enzymatic digestion,and endothelial cells were isolated by immunomagnetic beads sorting.Morphology and growth characteristics of two types of cells were observed with an inverted phase contrast microscope,their purity were identified by immunofluorescence,and their proliferation characteristics were observed by CCK-8 assay.At the level of cellular function,angiogenic capacity of endothelial cell was assessed by angiogenesis assay and smooth muscle cell responsiveness to platelet-derived growth factor(PDGF)was assessed by migration assay.Results The two types of cells isolated using this method grew vigorously and were in good condition.Smooth muscle cells exhibited typical“peak valley”growth,and immunofluorescence results showed cytoplasmic specific smooth muscleα-SMA and SM22αexpression was positive.Endothelial cells exhibited typical“cobblestone like”growth,with positive expression of platelet endothelial cell adhesion molecule CD31 and atresia zone protein 1.Conclusion This study established a reliable and efficient method for simultaneously isolating two types of cerebrovascular cells,the isolated cells have high purity,good activity,and stable characteristics after passage,which were sufficient to meet the needs of subsequent experiments.

关 键 词：脑血管内皮细胞 脑血管平滑肌细胞 葡聚糖梯度离心联合酶消化法 免疫磁珠分选 

分 类 号：R74[医药卫生—神经病学与精神病学] R5[医药卫生—临床医学]