The Primary Investigation Of CXCR7 And Its Ligand SDF-1 Mediation In Cell Migration

Cell migration is movement in response to migration signals or the increasing chemokine concentration towards the source of the chemokine. Chemokines, a kind of signal molecule, bind to cell surface receptor,through cell signaling pathway,active or inhibit the activity of actin binding protein,reorganize the cystoskeleton,cause cell migration. Stromal cell derived factor-1,SDF-1,is a significant chemokine,expressed in all kinds of cells and tissus, including immunological cell,brain,heart,kidney,liver,lung and spleen, has significant effect in development of immune system,circulation system and central nervous system.meanwhile,SDF-1 is correlate with HIV,inflammatory reaction,regulation of hematopoietic cell,migration of tumour cell.It is always considered that SDF-1 regulate all kinds of process throught CXCR4,but now a new receptor has be finded,named CXCR7.CXCR7 is expressed in many turmor cell,activated endodermis cell and embryo liver cell.In despit of CXCR7 not located in normal cells surface, mRNA of CXCR7 is finded in the cells.It is proved that CXCR7 promote cell survival in vitro,and tumor proliferation in mouse.but the effect for cell migration is not clear.Two experiment were included as follows:1.Establised the recombinant expression vector, pGEX-4T-1- SDF-1α,with pMD-18T-SDF-1αand pGEX-4T-1,The plasmid was trans-formed into E.coli BL21 and induced to express fusion protein with IPTG..The expression of GST-SDF-1αwas detected,and purified by Glutathione Sepharose 4B.2.Two peptides (H458,H459) were produced and conjugate with KLH.,the peptide-KLH was mix with Fredund's complete or incomplete adjuvant,and then used to immunize rabbits,Antibody was precipitated by ammonium sulphate saturation, Decte titer of antiserum by means of ELISA , Western-blot and indirect immunofluorescence.Use Millicell system to investigate the effect of SDF-1/CXCR7 on cell migration. Take count of the migrate cell from antibody group, chemokine group,and normal control group.RESULTS: Our successful cloning and expression of SDF-1αgene and purification GST-SDF-1αof protein, and was mostly in the form of inclusion bodies.The protein GST-SDF-1αwas purified by Glutathione Sepharose 4B. the peptide-KLH was used to immunize rabbits,the titer of antiserum was 1:8000.Western-blot analysis showed the antiserum could combine with disruption of Hela and MCF-7.Indirect immunofluorescence showed the masccline located in the cell surface of Hela and MCF-7.The number of migration cell is evident difference between antibody group and chemokine groupCONCLUSION: GST-SDF-1αwas successfully cloned and obtaianed expression product of fusion protein.Obtained the CXCR7 antiserum from rabbits.the antiserum inhibit cell migrate by SDF-1/CXCR7.Laid the foundation for further studies SDF-1/CXCR7 and the mechanism of cell migration...