Gene Cloning And Function Study On Megalobrama Amblycephala Macrophage Colony Stimulating Factor And Its Receptor

Macrophages are important immune cells,and macrophage colony stimulating factor(CSF-1)is a key regulator for the survival,proliferation and differentiation of macrophages.In this study,through the gene cloning,prokaryotic protein expression and specific antibody preparation of Megalobrama amblycephala macrophage colony stimulating factor(MaCSF-1)and its receptor(MaCSF-1R),the effects of MaCSF-1on proliferation,phagocytosis and secretion of inflammatory factors of monocytes/macrophages,as well as the mechanism were studied.The main research results are as follows:1.MaCSF-1 and MaCSF-1R gene cloning and molecular characteristics.The full-length cDNA of MaCSF-1(MW805228)contains 1626 nucleotides(nts),with a molecular weight of 60.3 k Da and an isoelectric point(p I)of 9.03.Similar to CSF-1 in mammals and other reported bony fishes,MaCSF-1 contains a signal peptide(located at aa positions 1-28),a CSF-1 domain(located at aa positions36-177),and two low-complexity region(located at aa positions 201-214 and222-233),a short transmembrane domain(located at aa positions 499-521),and has four highly conserved cysteine residues.By comparing amino acid sequences,MaCSF-1 was found to have high homology with CSF-1 of other reported bony fishes and mammals,and the highest homology with grass carp was 94.83%.MaCSF-1R cDNA(MW805230)is 2931 nucleotides(nts)in length,with a molecular weight(MW)of 110 k Da and an isoelectric point(p I)of 5.84.Except for one less IgG-like domain,MaCSF-1R is similar to CSF-1R in mammals and other reported teleost fishes,including a signal peptide(located at aa positions 1-17),four IgG-like domains(located at aa positions 32-110,122-205,224-295 and 325-412),a short transmembrane domain,and a cytoplasmic tyrosine kinase domain.By comparing amino acid sequences,MaCSF-1R showed high homology with other bony fishes and mammals,among which the homology with grass carp was 96.62%.q RT-PCR and WB results showed that under normal physiological conditions,MaCSF-1 expression was highest in spleen,followed by blood,head kidney,muscle,liver,brain,heart,intestine and gill.The expression of MaCSF-1R was highest in spleen,followed by head and kidney,blood,liver,muscle,intestine,brain,heart and gill.2.MaCSF-1 activates monocytes/macrophages.MaCSF-1 has the biological activity of activating monocytes/macrophages in the head kidney of M.amblycephala.The CCK-8 cell proliferation assay showed that after 300 ng/m L rMaCSF-1 treatment for 2 h,the proliferation rate was 11.1%compared with the control group.Flow cytometry analysis showed that the phagocytosis rate was more than 30% after 300 ng/m L rMaCSF-1 treatment for 4 h.The q TR-PCR results showed that the secretion of inflammatory factor IL-10 increased significantly at 4 h after 300 ng/m L rMaCSF-1 treatment,which was 6.4times that of PBS control group.3.MaCSF-1 activates monocytes/macrophages by binding to MaCSF-1R.Indirect immunofluorescence results showed that MaCSF-1R was localized on the macrophage membrane.AfterMaCSF-1R specific antibody blockage,the stimulating effects of MaCSF-1 on the proliferation,phagocytosis and IL-10 expression was significantly inhibited compared with the control group.Pull-down test results showed that rMaCSF-1 bound to rMaCSF-1R,and the indirect immunofluorescence results also showed that MaCSF-1 bound to MaCSF-1R in macrophages.4.MaCSF-1R can be used as a molecular marker on the surface of monocytes/macrophages,and MaCSF-1R Ab may be used to sort macrophages.According to previous studies,MaCSF-1R,CD11 b,CD4,CD8,TCRβ,IgM,IgD,and MPX were selected as cell surface molecular markers to identify monocytes/macrophages,T cells,B cells,and neutrophils of M.amblycephala.Semi-quantitative PCR results showed that the bands of CD4,CD8,TCRβ,IgM and IgD were bright in the freshly isolated head kindey single cell suspension,followed by MaCSF-1R and CD11 b,and finally MPX,indicating that the proportion of T cells and B cells in the freshly isolated head kindey single cell suspension was the highest,followed by monocytes/macrophages,and finally neutrophils.However,MaCSF-1R and CD11 b were bright,MPX bands were weak,CD4,CD8,TCRβ,IgM and IgD bands basically disappeared in the cells after 3 d adherent purification culture.MaCSF-1R Ab was used to label monocytes/macrophages,and the flow cytometry results showed that the positive rate of MaCSF-1R Ab was more than 10%.These results indicate that high purity macrophages can be obtained after purification and culture,MaCSF-1R can be used as a molecular marker on the surface of monocytes/macrophages,and MaCSF-1R Ab may be used to sort monocytes/macrophages.5.Monocytes/macrophages can be subcultured under rMaCSF-1 additionThe primary isolated monocytes/macrophages of the head kidney could be subcultured for 2 generations with the addition of rMaCSF-1.In the process of culture,it was found that the cells with rMaCSF-1 addition had a good adhesion effect,and it was easy to form colonies with adherent cells as the core and expanding continuously.In conclusion,MaCSF-1 has biological activities to promote macrophage proliferation,phagocytosis and cytokine secretion,and MaCSF-1R mediates MaCSF-1 for the activation of macrophages.In addition,MaCSF-1R can be used as a monocyte/macrophage specific marker and may be used for monocyte/macrophage sorting.This study provides a better understanding of the biological activities and relationships of MaCSF-1 and MaCSF-1R in teleost fish.