S1P1受体细胞模型的构建及其功能特性的初步研究

Preliminary study of functional characteristics of sphingosine 1-phosphate receptor type 1 with established cell model

目的构建1型1-磷酸鞘氨醇受体(S1P1)的细胞模型,初步探讨其介导的功能特性。方法用PolyFect,将包含全长人S1P1编码区与EGFP融合基因的载体HA-S1P1-Myc-EGFP-N1转入CHO细胞,经G418筛选,获得S1P1-EGFP-CHO稳定细胞株。以空载体pEGFP-N1转染的CHO稳定细胞株作为阴性对照,镜下观察细胞的形态。100nM FTY720处理S1P1-EGFP-CHO细胞5、24小时后用荧光显微镜观察S1P1在细胞上的表达。结果成功构建S1P1-EGFP-CHO细胞模型,表达S1P1的细胞变圆,FTY720导致该细胞模型的S1P1长时间内化。结论S1P1-EGFP-CHO细胞可作为S1P1功能特性研究的细胞模型,S1P1能使细胞变圆,FTY720能导致S1P1长时间内化。

Objective To establish sphingosine 1-phosphate receptor type 1（S1P1） cell model. Methods The eukaryotic expression vector HA-S1P1-Mye-EGFP-N1 containing the coding region of human full length S1P1 and EGFP fusion gene was transfected into CHO cells with PolyFect. The transfected CHO cells were selected with G418 and S1P1-EGFP-CHO ceils were obtained. With pEGFP-N1-transfeeted CHO （EGFP-CHO） cells as negative control, cell morphology was analyzed using an inverted microscope. S1P1-EGFP-CHO cells were incubated for 5 or 24 hours in the absence and presence of 100 nM FTY720, and the S1P1 expression was analyzed with fluorescence microscopy. Results S1P1-EGFP-CHO cell was successfully established. CHO ceils rounded with transfection of S1P1. FTY720 induced S1P1 long-term internalization. Conclusion A model of S1P1-EGFP-CHO cell is suecessesful established.