一种高纯度小鼠原代脑微血管内皮细胞的分离培养方法

Isolation and culture of primary microvascular endothelial cells from mouse brains

背景:脑微血管内皮细胞是神经科学研究领域的重要工具细胞,如何获得高纯度的脑微血管内皮细胞一直是体外研究的关键和难点。目的:探索一种简便易操作,高纯度的原代脑微血管内皮细胞的分离培养方法。方法:使用6-8周龄C57BL/6小鼠,经酶消化及梯度离心法获得微血管段,使用药物进一步纯化内皮细胞,利用CD31和GFAP免疫荧光染色鉴定内皮细胞纯度。使用Claudin-5免疫荧光染色鉴定紧密连接蛋白的表达情况。结果与结论:1细胞呈旋涡状或铺路石样排列、生长;2免疫荧光显示内皮细胞纯度达99%以上,并广泛表达紧密连接蛋白;3结果表明,实验建立了一种简便、易操作的,可以获得大量高纯度的原代小鼠脑微血管内皮细胞的培养方法。

BACKGROUND：Brain microvascular endothelial cel s （BMECs） are important tools in the field of neuroscience research;therefore, how to obtain highly purified BMECs is a key and difficulty in vitro. OBJECTIVE：To develop a simple method of isolating and culturing highly purified BMECs. METHODS：C57BL/6 mice aged 6-8 weeks old were selected, and microvessels were obtained using enzyme digestion and gradient centrifugation. Further, endothelia cel s were purified by certain drugs, fol owed by identified by CD31 and GFAP immunofluorescence staining. The expression of Claudin-5 was detected using immunofluorescence staining with anti-Claudin-5 antibody. RESULTS AND CONCLUSION：Mouse BMECs grew and arranged in spiral or cobblestone-like. Immunofluorescence staining showed that the purity of BMECs reached above 99%and Claudin-5 was highly expressed. In conclusion, a simple method of easy accessibility is developed to obtain highly purified primary mouse BMECs.