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. 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[J]. 心脏杂志, 2023, 35(2): 136-140. DOI: 10.12125/j.chj.202205063
    引用本文: 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. 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[J]. 心脏杂志, 2023, 35(2): 136-140. DOI: 10.12125/j.chj.202205063
    Mei-jie LIU, Jia-xin ZHANG, Jia-heng ZHOU, Jiong AN, Hao-miao BAI, Xu-hui HU, Pei-ling LI, Hong-yan YANG, Jia LI. Primary culture and characterization of microvascular endothelial cells from murine skeletal muscle with immuomagnetic beads[J]. Chinese Heart Journal, 2023, 35(2): 136-140. DOI: 10.12125/j.chj.202205063
    Citation: Mei-jie LIU, Jia-xin ZHANG, Jia-heng ZHOU, Jiong AN, Hao-miao BAI, Xu-hui HU, Pei-ling LI, Hong-yan YANG, Jia LI. Primary culture and characterization of microvascular endothelial cells from murine skeletal muscle with immuomagnetic beads[J]. Chinese Heart Journal, 2023, 35(2): 136-140. DOI: 10.12125/j.chj.202205063

    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

    • 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。

       

      Abstract:
        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.

       

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