在Transwell小室中分离培养人脐静脉内皮细胞的研究

Isolation and Culture of Human Umbilical Vein Endothelial Cells in Transwell Chamber

目的建立一种在特殊材料Transwell培养小室中分离培养原代人脐静脉内皮细胞的技术。方法采用酶消化法分离得到人脐静脉内皮细胞(HUVEC),免疫荧光法检测内皮因子Ⅷ予以鉴定,待长满后用不含EDTA的0.25%胰酶传代到Transwell小室中,观察小室中HUVEC的生长情况。结果在脐带长度一定的情况下,HUVEC分离所得到细胞的多少以及细胞的状态与胰酶的用量、时间、温度及是否含EDTA关系密切。本研究胰酶消化时间在室温(25℃左右)情况下要消化30min,用的胰酶是含EDTA和不含EDTA的胰酶1∶1混合;长满之后传代消化的胰酶只能用不含EDTA的胰酶消化,时间以在镜下的细胞状态为准。结论在特殊材料Transwell小室中成功分离培养HUVEC,一定要在比较温和的条件下消化HUVEC,这样才可能得到活力比较好、数量比较多的细胞。

Objective To establish a method of primary human umbilical vein endothelial cell separation and cuhure in special materials Transwell. Methods Human umbilical vein endothelial cells （HUVEC） were isolated by enzyme digestion, endothelial factor VIII was identified by immunofluorescence; the HUVEC cell was passaged to Transwell chamber by 0.25% trypsin free of EDTA when it was full in the glass bottle and the cell vitality was observed in the chamber. Results When umbilical cord length is equal, the quantity and vitality of HUVEC cells were relative to dosage, time, and temperature and whether or not containing EDTA. In microscope. Conclusion The successful isolation of HUVEC in special materials Transwell suggests that digestion condition of HUVEC was the key for the quantity and vitality of HUVEC. In mild conditions, it may get better, more vitality of cells.