一种大鼠主动脉内皮细胞分离培养的改良方法

An Improved Method of Isolating and Cultivating of Aortic Endothelial Cells in Rats

目的探索一套原代培养大鼠主动脉内皮细胞(RAEC)简单实用的方法体系。方法 SD大鼠两只,无菌环境下分离胸腹主动脉,清除脂肪、结缔组织。显微剪剪开主动脉,内膜面向下贴附于少量Ⅰ型胶原酶上消化60min。离心收集内皮细胞置于1%明胶包被的六孔板,加入20%胎牛血清的M199中培养。3～4天后换液更换内皮细胞培养基(ECM)培养。当细胞长满约90%底面积时,玻璃探针机械剔除成纤维样细胞后传代。倒置显微镜和抗Ⅷ因子抗体对细胞分别做形态学和免疫细胞化学鉴定。结果原代培养3～4天时,少量细胞开始贴壁并分裂生长。更换ECM后细胞增殖迅速,培养8～10天时可见细胞长满90%左右的底面积,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内皮细胞中有特定Ⅷ因子表达。原代周期8～10天,传代周期3～4天,经纯化传代后纯度接近100%。传至第6代仍未见细胞分裂增殖活性下降。结论本实验体系培养RAEC简单有效,重复性良好。获得细胞数量多、纯度高,有利于实验人员掌握。

Objective To explore a set of simple and effective system which cultures primary rat aortic endothelial cells （ RAEC）. Methods The thoracic aorta and abdominal aortas were get under sterile conditions from two SD rrats. The peripheral fats and connective tissues were removed. The aorta was cut with microsurgical scissors.. The inner face was put down on a small amount of collagenase type I solution and then digested for 60 minutes. EC should be collected by centrifugation at 1000r/min for 5 minutes. Then the cells were gently added with 2.5ml of 20% FBS - M199 and cultured in a gelatin - coated muhiwell plate. After 3 -4 days incubation at 37℃ , the medium M199 was removed, and medium ECM was added. When the cells were covered with about 90% bottom area, we used glass probe to knockout fibroblasts and then passaged them. Inverted microscope and anti - Ⅷ factor antibody were used to identify the morpho- logical and immunocytochemistry characteristics. Results After 3 - 4 days, we could see that a small number of cells were attached to the bottom of the plate and begun to split. The cells grew rapidly after being replaced with the medium ECM. 8 - 10 days later, the migrating cells covered about 90% of the bottom area, and they showed a typical ＂ paving stone＂ or ＂ cobblestone＂ appearance. The immuno- cytochemistry study demonstrated specific expression of factor Ⅷ in isolated EC cultures. The growth cycle of primary RAEC were 8 - 10 days and passage cycle was 3 -4 days. After it was purified and subcultured, the cells purity reached almost 100%. The primary cells could be passaged at least six times without proliferation activity declined. Conclusion We have developed a system to isolate and culture EC from rat aorta that is simple, effective and easy to replicate. It is easy for us to handle, and at the same time the ceils have high quality and quantity.