人脐静脉内皮细胞的培养及鉴定

Culture and identification of human endothelial cells derived from umbilical veins

目的 探讨新生儿脐静脉内皮细胞的培养及鉴定方法 ,建立血管内皮细胞培养模型 ,为体外研究血管内皮细胞提供实验手段。方法 采用胶原酶Ⅱ灌流消化法培养人脐静脉内皮细胞 ,当原代培养细胞 80 %以上汇合后 ,用胰蛋白酶消化传代 ;根据细胞生长特点、形态特征、细胞表型和分泌蛋白流式细胞术 (FM术 )免疫荧光检查对细胞进行鉴定。结果 种植在培养瓶中的内皮细胞 12小时贴壁生长 ,48～ 72小时生长最快 ,7～ 10天汇合。内皮细胞呈接触抑制生长、呈鹅卵石样外观 ,FM术检测CD3 1和VⅢ -R -Ag均为阳性表达。 结论 消化酶灌注脐静脉消化内皮细胞是获取血管内皮细胞的一种好方法 ,可靠性大 ,成功率高 。

Objective To extablish the methods of culturing human endothelial cells (EC) and identifing the cells according to the antigens expressed and their morphological aspect. Methods After digestion with Ⅱ type collagenase at 37℃ for 10 minutes, the cells were centrifuged, and the cell pellet was resuspended in DEME medium containing fetal bovine serum (10% vol/vol), seeded on gelatin(0.1% wt/vol) procoated dishes at aconcentration of 10,000 per square centimeter. The medium was changed 1 days later when the cells were attached and every 2 days, until the cells reached confluence. Phase contrast microscopy and flow cytometry with specific antisera against von willebrand factor and CD31 were used to observe and identify the culturedcells. Results At confuluence(7～9 days), the cultured cells had a cobbestone appearance with a strict monolayer growth and contact inhibition. The mean fluorescence intensity of the cells was similar with both antibodies for the cultured cells vs controls. Conclusion The cultured cells are endothelial cells, and the method of identification is practical and convenient.