成年大鼠心肌微血管内皮细胞的分离与培养

Isolation and culture of adult rat cardiac microvascular endothelial cells

目的本文建立一种简便、高效的成年大鼠心肌微血管内皮细胞分离及培养的方法。方法选取7~8周龄Wistar大鼠,麻醉后开胸取出心脏,采用离体心脏灌流和酶消化法进行心肌微血管内皮细胞的分离;取第3代细胞观察其增殖特征并绘制增殖曲线;用免疫细胞化学和免疫荧光对细胞的第Ⅷ因子相关抗原和CD31进行鉴定,利用基质胶检验其血管形成能力。结果体外培养心肌微血管内皮细胞呈多边形或梭形,具有典型的铺路石样生长特点;同时,第Ⅷ因子相关抗原和CD31呈阳性;具备血管形成能力。结论本方法培养的成年大鼠心肌微血管内皮细胞具有较高的纯度,方法简便易行,可用于心血管疾病基础及临床研究。

Objective To establish a simple and efficient method for isolation and culture of adult rat cardiac microvascular endothelial cells.Methods Wistar rats were anesthetized and thoracotomy was performed.Cardiac microvascular endothelial cells were isolated by heart perfusion and enzyme digestion.The morphology and growth curve of the third generation cells were examined by inverted microscopy.Its molecular markers factorⅧ-related antigen and CD31 were detected by immunocytochemistry and immunofluoresence.The angiogenic ability was examined by matrigel.Results The primary cultured cardiac microvascular endothelial cells in vitro were polygonal or fusiform with typical paving stone-like growth characteristics.At the same time,the cells were positive stained by factorⅧ-related antigen and CD31 and showed angiogenic ability.Conclusions Cardiac microvascular endothelial cells cultured by this method possess high purity.The method is easy to operate and can be used for laboratory and clinical research of cardiovascular diseases.