| P2Y1 receptor belongs to the family A of G protein-coupled receptors and plays an important role in thrombotic disorders.It will lead to a short time of platelet deformation and aggregation when it was activated.It was considered to be the necessary common factors to platelet activation and a potential target for the treatment of thrombotic disorders.Currently,nucleotide derivative MRS2179,MRS2500 have benn proved with anti-thrombotic activity in vivo and in vitro,they are the most common tools for P2Y1 receptor drug research,but there are bad inoral bioavailability.In this study,the P2Y1 was labled with the green fluorescent protein(GFP)through direct or indirect method and transfected into U2OS cell to build stable cell lines.These cell lines will bring more benefit in developing of drug or screening leading compound from natural products.At first,three eukaryotic expression vectors including beta inhibiting protein 2(beta arrestin2)tagged with GFP,P2Y1 receptor with hygromycin resistance and P2Y1 recrptor directly labled with GFP were constructed.Those vectors have been introducted into the U2OS cel by transinfection.Two stable cell lines have been established after selected by resistance screening and flow cytometry sorting.One cell line has a directly GFP-labled P2Y1 expression and the other have a indirectly GFP-labled P2Y1 expression.High level of target protein expressed in cell line was confirmed by Western-bloting.Receptor internalization and the aggregation of fluorescent spots can be observed in two kinds of cell lines after agonists ADP was added in culture medium.Analysis from high content system showed that two kind of cell lines’s fluorescence would strengthen after treating with ADP,and the intensity of fluorescence has a dose effect relationship of agonist.Under the condition of ADP concentration at 1.03 x 10-5 mol/L,and the effect time of 15 min at room temperature,the indirect marked cell line showed the highest average fluorescence intensity.Under the condition of the ADP concentration at 1.03x10-4 mol/L,and the effect time of 15 min at room temperature,the direct marked cell line also showed the highest average fluorescence intensity.The function of labled P2Y1 cell lines can be inhibited by P2Y1 antagonists of MRS2179 at concentration of 1x10-6 mol/L.In this study,two P2Y1 recptor labled by direct and indirect GFP cell lines were established.The function of P2Y1 quantitative assay in both cell lines was confirmed by aggregation of fluorescent spots under high content system.The detection method is easy to be standardized,and has good repeatability and high specificity characteristics.Direct and indirect P2Y1 labled cell lines have been considered as an ideal model to perform high-throughput screening of P2Y1 ligand in natural compound from traditional Chinese herb. |
