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- 2018
大鼠主动脉内皮细胞改良型分离培养方法
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Abstract:
摘要：目的 建立一种简便的大鼠主动脉血管内皮细胞(vascular endothelial cells, VECs)分离纯化及培养方法。方法 无菌条件下分离获得大鼠主动脉，在眼科显微器械的辅助下翻转暴露血管内膜。按处理方式的不同，将获得的主动脉血管段分为消化压片法组、单纯消化法组和单纯压片法组，并进行对应的处理和培养，通过倒置相差显微镜观察VECs形态，免疫荧光法鉴定VECs的细胞标记物。结果 消化压片法组在接种后48～72h即有VECs自血管段中迁移出，原代细胞约10～11d可达到近融合状态，子代细胞生长速度加快，5d后达到融合状态，呈典型的“铺路石”样特征。相对于其他两组，消化压片法组VECs的迁移速度快、细胞数量多。免疫学鉴定显示细胞标志物Ⅷ因子、vWF和CD31的阳性率高达99%。所培养的VECs传代培养至第10代，未见明显的细胞形态特征变化，免疫荧光鉴定至第8代，未见明显的细胞免疫特征变化。结论 本研究成功建立了一种简单经济的大鼠主动脉VECs分离、纯化和培养方法。
ABSTRACT: Objective To establish a simple method of isolating, purifying and cultivating vascular endothelial cells (VECs) of rat aortas. Methods The aortas of rats were isolated and obtained under aseptic conditions. The intima of aortas were turned over and exposed using ophthalmic microscopic surgical instruments. The fragments of the aortas were divided into digesting-sheeting group (DSG), digesting group (DG), and sheeting group (SG) based on the different process modes, and then received cultivation. Morphological characterization was performed using phase contrast microscopy; further characterization was analyzed by immunofluorescence method. Results VECs migrated out of the fragments of aortas after 48-72 hours of plating, and the primary cells reached subconfluence on days 10-11. Their subcultures grew faster than the primary and reached confluence on day 5. The cells and their subpassages showed VECs’ morphology with typical cobblestone appearance. The cells of DSG migrated from the fragments of aortas faster than those of DG and SG, and the quantity of primary cells of DSG were larger than those of DG and SG. Ninety-nine percent of the cell population had positive staining for factor VIII, von Willebrand factor and CD31. VECs cultured in this study could be passaged repeatedly for 10 passages without significant loss of typical features, and immunologically identified at the 8th generation without features changed. Conclusion We have developed a simple and economic method to isolate, purify and culture VECs of rat aortas
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