人脐静脉内皮细胞1-磷酸鞘氨醇受体鉴定及其应用探讨

Identification and application of sphingosine-1-phosphate receptor of human umbilical vein endothelial cells

目的鉴定人脐静脉内皮融合细胞株EA.hy926 和人脐静脉内皮细胞（HUVEC）CRL-1730 1-磷酸鞘氨醇受体（S1PR）亚型和胞浆中C-myc 及His 标签蛋白的表达情况,为研究载脂蛋白M（ApoM）-1-磷酸鞘氨醇轴（ApoMS1P轴）的功能提供参考.方法体外培养EA.hy926 及CRL-1730 至细胞密度达到60%～70%时,采用细胞免疫荧光技术鉴定内皮细胞标志凝血八因子（FⅧ）、ApoM、S1PR1-S1PR5、C-myc 和His 标签蛋白.结果（1）两种细胞均表达FⅧ和ApoM,FⅧ在CRL-1730 中呈散在颗粒状分布,在EA.hy926 中呈均匀分布.（2）两种细胞均主要表达S1PR1,少量表达S1PR2 和S1PR3,不表达S1PR4 和S1PR5.（3）两种细胞胞浆中均有C-myc 和His 标签蛋白的表达.结论两种细胞都具有内皮细胞的特性;因其自身表达ApoM、C-myc 和His 标签蛋白,在应用这两种细胞研究ApoM-S1P 轴的功能时,不适合选用带有C-myc 和（或）His 标签的重组ApoM 蛋白.

Objective To identify the expression of sphingosine-1-phosphate receptor (S1PR) subtypes, C-myc and His tag proteins of human umbilical vein endothelial fusion cell line, EA.hy926 and human umbilical vein endothelial cells (HUVEC), CRL-1730 for studying the function of apolipoprotein M (ApoM)-S1P axis. Methods Two kinds of cells (EA. hy926 and CRL-1730) were cultured to reach the density of 60%-70% in vitro. Immunofluorescence technique was em?ployed to investigate the expressions of coagulation factorⅧ(FⅧ), ApoM, S1PR1-S1PR5, C-myc and His tag proteins. Re?sults (1) Two kinds of cells both expressed FⅧand ApoM. FⅧpresented scattered particle distribution in CRL-1730, while uniform distribution in EA.hy926. However, ApoM was strongly expressed and widely distributed in cytoplasm of two kinds of cells. (2) S1PR1-3 can be detected on their membrane other than S1PR4 and S1PR5. S1PR1 was highly expressed but S1PR2 and S1PR3 were in a low level expression. (3) Two kinds of cells both expressed C-myc and His tag proteins in cytoplasm. Conclusion Two kinds of cells have the properties of endothelial cells and can express FⅧ, ApoM, C-myc and His tag proteins. It is not suitable for choosing C-myc and/or His tag–conjugated recombinant ApoM to study the fuction of ApoM-S1P axis with these two kinds of cells.