原代小鼠肺微血管内皮细胞的培养及鉴定

Primary culture and identification of mouse pulmonary microvascular endothelial cells

目的建立一种简单高效的小鼠肺微血管内皮细胞原代培养方法。方法选取2~3周ICR小鼠,剪开胸腹腔,取距离肺边缘约1.5mm组织,剪碎成米粒状,置于含有培养液的离心管中,离心洗涤后种瓶进行原代培养。通过细胞形态学观察、细胞Ⅷ因子相关抗原免疫细胞化学染色鉴定所培养的细胞。结果接种24h后,红细胞从贴壁的肺组织块边缘向四周游离;48h后,肺微血管内皮细胞爬出,单个细胞形态为多角形或短梭形,细胞间隙较大,胞核清晰,胞浆丰富;96h后细胞融合,呈典型的单层、铺路石样镶嵌式排列。细胞Ⅷ因子免疫细胞化学染色检测,胞质呈棕红色,表达为阳性,阳性细胞率达98%以上。结论随机组织块法能够成功高效分离培养出原代小鼠肺微血管内皮细胞。

Objective To establish a simple and efficient method for primary culture and identification of mouse pulmonary microvascular endothelial cells.Methods Select 2~3-week-old ICR mice,open the thoracic and abdominal cavity with scissors,take the tissue about 1.5 mm away from the edge of lung,cut it into rice grain-shaped pieces,put them in the centrifuge tube containing the culture medium and wash,then centrifuge and seed them in the bottles for primary culture.The cultured cells were identified by morphological observation and immunocytochemical staining of factor Ⅷ related antigen.Results After 24 hrs of seeding,the erythrocytes dissociated from the edge of the adherent pulmonary tissue block to the surroundings;48 hrs later,the pulmonary microvascular endothelial cells crawled out,and the morphology of every single cell was polygonal or short spindle,with large intercellular space,clear nucleus and rich cytoplasm;96 hrs later,the cells reached confluence and arranged in a typical monolayer,paving stone like mosaic pattern.Immunocytochemical staining of factor Ⅷ showed the cytoplasm was brownish red and positive expressed,the positive rate was over 98%.Conclusion The primary generation of pulmonary microvascular endothelial cells can be successfully isolated and cultured by random tissue block method.