The Experimental Research About CLEC9A On Effects Of Features And Functions Of Dendritic Cells

Objective:Transfect of CLEC9 A plasmid by Lipofectamin to mouse dendritic cells and dendritic cell line DC2.4,construct DC/CLEC9 A and DC2.4/CLEC9 A with higher expression of CLEC9 A,and then detect the effect and features of DC/CLEC9 A and DC2.4/CLEC9A。Methods:Femur bone marrow cells were extracted from 4-8-week-old male SD rats, then screening RBC by red blood cell lysate,finally we can obtain bone marrow-mononuclear cells,(BM-MNCs), in which the adherent cells were cultured in the medium with the cytokine GM-CSF, IL-4, and TNF-α and they become mature, observe their morphological changes daily; dendritic cell line DC2.4 to be transfected were kindly given by Second Affiliated Hospital of Soochow University. Collected the mature DC and DC2.4 and then transfected with different concentrations plasmid- liposome complexes, fluorescence and Real-Time PCR were used to find out the optimal transfection concentration; CLEC9 A was transfected into dendritic cells and DC2.4(DC/CLEC9 A and DC2.4/CLEC9A),nontransfected and blank vector CLEC9A-transfected dendritic cells were used as controls, Flow cytometry was used to detect their transfection efficiency, PCR and Western blot assay were used to detect expression of CLEC9 A on DC and DC2.4 cells,finally,we can construct DC/CLEC9 A and DC2.4/CLEC9 A with higher expression of CLEC9 A.The next next step is to detect the expression of surface molecules CD80, CD83, CD86 and MHCⅡby PCR and flow cytometry after tumor antigen stimulation,then research the function and feature of DC before and after transfection.Results:Transfecting rat bone marrow DC and DC2.4 cells by different concentrations of plasmid- liposome complexes,we found that the best concentration of plasmid CLEC9 A is 3ug, That the proportion of liposome and plasmid is 3ul and 1ug has the best transfection efficiency.After transfection, expression of CLEC9 A on rat bone marrow DC and DC2.4 cell surface were both higher than non-transfected and blank vector cells, compared to bone marrow DC, transfection efficiency of DC2.4 cells were significantly improved, about 2-fold increase. After the stimulation of rat tumor antigens, the expression of cell surface molecule CD80, CD83, CD86 and MHC Ⅱ on DC / CLEC9 A increased significantly compared with non-transfected and blank vector cells DCs, but expression of CD80, CD83, CD86 and MHCⅡon DC2.4 / CLEC9 A cell was not detected increased compared with nontransfected and blank vector DC2.4.Conclusion: 1. The transfection efficiency of DC2.4 cells grown adherent was significantly higher thanthe DC cell which growth of semi-suspended state by LipofectamineTM2000. 2. The proportion of CLEC9 A plasmid and LipofectamineTM2000(3 μg: 9 μL)is the bestconcentration for transfection 3. DC2.4 cell lines grow in adherent state, is lack of surface molecule expression, such asCD80, CD83, CD86 and MHCⅡwhich is normally expressed on DC cells, althoughthey has a higher transfection efficiency, the expression of surface molecules did nothave a corresponding significant increase after the tumor antigen. DC2.4 cell linepossible gradually lost normal DC cell activity and physiological functions during thegeneration grow; 4. The higher expression of CLEC9 A on rat bone marrow DC can enhance the activity andfunction of DC cells, it has certain clinical application.