改良大鼠心脏微血管内皮细胞的培养方法及鉴定

An improved culture method of isolating and characterizing cardiac microvascular endothelial cells in rats

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 kind of simple and effective system which cultures primary rat cardiac microvascular endothelial cells. Methods The hearts were rapidly obtained under sterile conditions from two Sprague Dawley rats （ one month~ old, about 100g weight） and the outer one -fourth of the left ventricular tissue was removed. The remaining heart tissue was minced finely, incubated with collagenase and trypsin. Cells should be collected by centrifugation after filtered. Then, the cells were suspended and plated on a T25 flask for 2 hours. Results Most cells were adhered to the bottom of the flask after 1 -2 h of plating, and began to split at 24 h. After 3 days of plating, the cells were 80% ~90% confluent and showed a typical paving stone or cobblestone appearance. The immunocytochemistry analysis showed that the adherent cells were positive for VIII factor and CD31. Moreover, the cultured cells could form tube at Matrigel in vitro. The percentage of P2 cells positive for VIII factor and CD31 was more than 95%. The growth cycle of primary RCMECs were 3 - 4 days and passage cycle was 2 - 3 days. The primary cells could be passed at least four times without morphological alteration and proliferation activity decline. Conclusion We have improved a system to isolate and culture RCMECs, which is easy to handle and replicate. Moreover, we could obtain more RCMECs with higher purity and increased activity.