目的建立一种分离大鼠心脏微血管内皮细胞的方法。方法雄性SD大鼠2只,快速取出心脏,去除左室外约1/4层,充分剪碎剩余组织,Ⅱ型胶原酶和胰酶先后消化20~30 min,过滤并离心收集细胞,加入培养液置于细胞培养箱中培养。结果原代细胞基本于接种后1~2 h已大部分贴壁,24 h细胞充分伸展,并进入对数生长期,细胞增殖迅速,培养3 d时,可见细胞汇合度达80%~90%,细胞长至融合后呈典型的“铺路石”或“鹅卵石”征。免疫细胞化学鉴定表明在分离培养的大鼠心脏微血管内皮细胞中有特定Ⅷ因子及CD31表达,体外小管形成实验证明大鼠心脏微血管内皮细胞具有小管形成的能力。原代周期3~4 d,传代周期2~3 d,P2代细胞纯度达95%以上。传至P4代仍未见细胞形态及分裂增殖活性下降。结论本方法培养大鼠心脏微血管内皮细胞简单有效,重复性好,且获得细胞数量多、纯度高,有利于实验人员掌握。 Objective To establish a method for isolation of rat cardiac microvascular endothelial cells. Methods Two male Sprague-Dawley rats were randomly divided into four groups: one-fourth of the left ventricle was removed, and the remaining tissue was harvested. The collagenase and trypsin were digested for 20-30 min, and the cells were collected by filtration and centrifugation. Placed in a cell culture incubator. Results The primary cells almost adhered to the surface 1 ~ 2 h after inoculation, 24 h cells were fully extended and entered the logarithmic growth phase, the cell proliferation was rapid. After cultured for 3 days, the cell confluency reached 80% -90% , Cells grow to the typical “paving stone” or “pebble” sign after fusion. Immunocytochemistry showed that specific factor Ⅷ and CD31 were expressed in isolated rat cardiac microvascular endothelial cells. In vitro tubular formation assay demonstrated the ability of rat cardiac microvascular endothelial cells to form tubules. The primary cycle of 3 ~ 4 d, passage cycle 2 ~ 3 d, P2 generation cell purity of more than 95%. It has not yet been seen in P4 generation that cell morphology and mitotic activity decrease. Conclusion The method of rat cardiac microvascular endothelial cells cultured simple and effective, good repeatability, and access to a large number of cells, high purity, is conducive to experimental personnel to master.