Study on relationship between conserved motif of sphingosine 1-phosphate receptor type 1 and FTY720-induced internalization / 中国免疫学杂志

Objective:To study the relation between conserved motif of sphingosine 1-phosphate receptor type 1(S1P1) and FTY720-induced internalization.Methods:With HA-S1P1(WT)-Myc-EGFP-N1 fusion vector as template,HA-S1P1(R142N)-Myc-EGFP-N1 fusion vector was constructed by overlap PCR.The conserved ERY motif of wild type S1P1 was mutated into ENY.The recombinant vectors were confirmed by sequencing,then they were transfected into HEK293 cells by Polyfect.The transfected HEK293 cells were selected with G418.Cells were incubated for 3,6,12 hours in the absence or presence of 100 nmol/L FTY720,then S1P1 gene expression was analyzed by fluorescence microscopy.Results:Sequencing confirmed HA-S1P1(R142N)-Myc-EGFP-N1 vectors was successfully constructed.S1P1(WT) protein and S1P1(R142N) protein were expressed on the stably transfected-HEK293 cell surface.FTY720 induced S1P1(WT) internalization,but not S1P1(R142N).Conclusion:FTY720-induced S1P1 internalization is related with conserved ERY motif.

Construction of the eukaryotic expressing vector of PcDNA3.1 rhGM-CSF and its effect on the proliferation and differentiation of K562 cells / 中国免疫学杂志

Objective:To construct eukaryotic expressing vector of PcDNA3.1-rhGM-CSF and to investigate its effects of suppressing growth and inducing differentiation in K562 cells.Methods:The eukaryotic expressing vector PcDNA3.1-hGM-CSF was constructed by means of PCR and T-A clone techniques as well as directional cloning techniques.It was affirmed by the restriction map and DNA sequence analysis,and then transfected into K562 cells.It was observed that the target gene of the recombinant vector was expressed and exerted in K562 cells 72 h later,RT-PCR was used to affirm the expression of rhGM-CSF in K562 cells;Cell morphological,cell proliferation assay and immunohistochemistry were used to observe the effect of PcDNA3.1-rhGM-CSF on the growth and differentiation of K562 cells.Results:①The recombinant eukaryotic expressing vector PcDNA3.1-GM-CSF was constructed successfully;②Recombinant GM-CSF was expressed in the transfected K562 cells and the transfected K562 cells could differentiate into monocyte and macrophage cells;③ The proliferation of K562 cells was suppressed.Conclusion:The recombinant eukaryotic expression vector PcDNA3.1-rhGM-CSF was constructed successfully;Transfected K562 cell could differentiate into monocyte and macrophage cell.