人脐静脉内皮细胞的原代培养及体外层流体系的建立

PRIMARY CULTURE OF ENDOTHELIAL CELLS DERIVED FROM UMBILICAL VEINS AND ESTABLISHMENT OF EXTRACORPOREALLAMINAR FOLW SYSTEM

目的:建立体外培养原代人脐静脉内皮细胞的方法及体外模拟自然生理剪切力条件下白细胞流动体系.方法:采用胶原酶、胰蛋白酶联合消化法培养人脐静脉内皮细胞,根据细胞生长特点、形态特征、免疫荧光对细胞进行鉴定,并通过体外层流体系观察白细胞和人脐静脉内皮细胞的相互作用.结果:种植在培养瓶中的内皮细胞4h贴壁生长,48～72 h生长最快,5～7 d汇合.内皮细胞呈接触抑制生长、呈鹅卵石样外观,VWF相关抗原检测为阳性.人脐静脉内皮细胞经凝血酶刺激后,内皮细胞表面表达P-选择素并可以介导白细胞在其表面滚动.讨论:形态学及免疫学鉴定结果表明培养细胞为人脐静脉内皮细胞,体外层流体系的建立为以后研究工作奠定了基础.

Objective:To establish a method of culturing Primary endothelial cells of human umbillical vein ;To mimic a leucocyte laminar flow system under a natural shearing force condition.Methods:Endothelial cells of human umbillical vein were digested with collagenase and trypsinase; Endothelial cells were identified according to the cell growth characteristic, morphological feature and immunofluorescence.; the interaction between the leucocytes and endothelial cells was observed with the extracorporeal laminar flow system.Results:Endothelial cells in the cultrre flask began their adhenent culture After 4h planting, the fastest growth occurred after 48～72h planting and confluency appeared after 5～7d planting. Endothelial cells growed in a pattern of contact inhibition ,showing a cobblestone-like appearance; VWF Relative Antigen Determination of endothelial cells was positive, surfaces of endothelial cells expressed P-selectin which mediated leucocytes rolling on it after stimulated by thrombin.Conclusion: The identified results show the culture cells are the endothelial cells of human umbillical vein ; establishment of leucocyte laminar flow system provides a basis for the series research.