免疫磁珠法分离培养小鼠原代骨骼肌微血管内皮细胞及鉴定  

Primary culture and characterization of microvascular endothelial cells from murine skeletal muscle with immuomagnetic beads

作　　者：刘美杰 张佳欣 周嘉恒 安冏 白浩淼 胡旭晖 李沛凌 杨红燕 李嘉 LIU Mei-jie;ZHANG Jia-xin;ZHOU Jia-heng;AN Jiong;BAI Hao-miao;HU Xu-hui;LI Pei-ling;YANG Hong-yan;LI Jia(Department of Rehabilitation Sciences,School of Aerospace Medicine,Air Force Medical University,Xi’an 710032,Shaanxi,China;Cadet Regiment,School of Basic Medical Sciences,Air Force Medical University,Xi’an 710032,Shaanxi,China)

机构地区：[1]空军军医大学航空航天医学系飞行人员疗养与康复教研室,陕西西安710032 [2]空军军医大学基础医学院学员队,陕西西安710032

出　　处：《心脏杂志》2023年第2期136-140,共5页Chinese Heart Journal

基　　金：国家自然科学基金项目(31771265,32071108);空军军医大学人才支持计划项目(2019XC089);空军军医大学重大问题科技攻关项目(2019ZTA06)。

摘　　要：目的旨在建立一种有效分离、培养、扩增小鼠原代骨骼肌微血管内皮细胞(MMECs)的方法。方法采用胶原酶消化法、差速贴壁法以及免疫磁珠法分离纯化骨骼肌微血管内皮细胞,贴壁培养进行体外扩增。利用相差显微镜观察培养细胞的形态、MTT法测定细胞的生长情况、CD31免疫荧光染色对其表型进行鉴定、流式细胞术鉴定细胞纯度。结果实验成功获取小鼠骨骼肌微血管内皮细胞,小鼠原代骨骼肌微血管内皮细胞在培养24 h后迅速贴壁,(3~6)d内迅速生长,呈梭形,单层排列,培养(6~8)d可长满培养皿底,呈铺路石样排布。MTT法测得培养第2代小鼠骨骼肌微血管内皮细胞的生长曲线呈倒“S”形。流式细胞术检测经差速贴壁联合磁珠纯化的CD31+的MMECs细胞可达97%以上。结论成功建立了一种简单、高效的分离小鼠骨骼肌微血管内皮细胞的方法,为开展与骨骼肌微血管内皮细胞相关研究提供了良好的实验方法和技术手段。AIM To establish an accurate and effective technique and method of isolating and culturing mouse skeletal muscle microvascular endothelial cells(MMECs)in vitro.METHODS Mouse MMECs were isolated and purified by collagenase I digesting and differential adhesion combined with magnetic beads and were adherently cultured in vitro.The cell morphology and ultrastructure were observed by microscopes,the growth curve of the cultured MMECs was measured by MTT,its phenotype was identified by CD31 related antigen immunofluorescence staining,and the purity of the MMECs was detected by flow cytometry.RESULTS Mouse skeletal muscle microvascular endothelial cells were obtained successfully and the primary cultured endothelial cells quickly adhered to the wall after 24 hours of culture,grew actively in monolayer with the characteristics of endothelial cell shape at(3~6)days of culture and fully covered the bottom in a“cobblestone”arrangement after(6~8)days of culture.MTT assay showed that cell growth curves of 2 generations of MMECs presented the inverted“S”shape.The results of flow cytometry indicated that the positive rate of CD31+labelled MMECs purified with differential adhesion plus magnetic beads was over 97%.CONCLUSION A simple,reproducible and well tested method is established for isolating microvascular cells of murine skeletal muscle,which can provide the material basis and experimental model for further research on microvascular-related myopathy and molecular biology in related fields.

关 键 词：细胞分离 原代培养 骨骼肌微血管内皮细胞 肌病 免疫磁珠 

分 类 号：R361[医药卫生—病理学]