改良贴壁法结合内皮细胞培养基培养小鼠肺微血管内皮细胞*☆

背景：肺微血管内皮细胞是研究微循环的重要内皮细胞模型之一，众多培养方法中单纯贴壁法操作相对简便，但耗时长，杂质细胞多，是批量培养细胞的最大障碍。 目的：建立优化的小鼠肺微血管内皮细胞体外培养方案，观察细胞生长状态并鉴定细胞性质。 方法：无菌状态下快速剪碎5日龄C57BL/6J小鼠的肺叶外周组织，肺组织颗粒贴壁法获得肺微血管内皮细胞，并用内皮细胞培养基培养。倒置显微镜观察培养细胞生长和行为状态，Ⅷ因子相关抗原免疫组化和免疫荧光进行细胞鉴定。 结果与结论：肺组织块培养24 h内可见梭形细胞爬出，传代后细胞生长迅速，形态规则呈鹅卵石状，纯度高达98%以上，结合Ⅷ因子相关抗原检测证实其为内皮细胞。结果可见联合运用肺组织颗粒贴壁和内皮细胞培养基可高效获得原代小鼠肺微血管内皮细胞。关键词：肺微血管内皮细胞；细胞培养；优化；鉴定；小鼠；血管组织工程 缩略语注释：PMVECs: pulmonary microvascular endothelial cells，肺微血管内皮细胞 doi:10.3969/j.issn.1673-8225.2012.15.007

Primary cultivation of mouse pulmonary microvascular endothelial cells by improved adherence method combined with endothelial cell culture medium

BACKGROUND: Pulmonary microvascular endothelial cells (PMVECs) are one of the most important endothelial cell models for microcirculation. During many training methods, simple adherence method is relatively simple, but the time-consuming and more impurity cells are the biggest obstacle for cells culture. OBJECTIVE: To optimize the cultivation method for mouse PMVECs in vitro, and to observe the growth condition and to identify the property. METHODS: The pulmonary tissue particles were isolated from 5-day-old C57BL/6J mice pups by scissoring peripheral lung lobes accurately under sterile conditions, and cultured with endothelial cell medium. The morphology and growing characters of the cells were observed by inverted microscope. Cultured cells were identified by immunohistochemistry and immunofluorescence staining of factor Ⅷ related antigen. RESULTS AND CONCLUSION: A few spindle-shaped cells were found along the edge of tissue particles after cultured for 24 hours. After subculturing, cells spread even faster and took on a homogeneous cobblestone-like appearance, with the purity exceeding 98%. Morphological observations and detections of Ⅷ related antigen results conformed that these cultured cells were endothelial cells. It is a convenient and effective approach to gain a large scale of PMVECs from neonatal mice by tissue-anchoring method and endothelial cell medium.