The Research Of Mice Bone Marrow-derived DC Extraction And Its’ Fusion Cells With Malignant Melanoma Cells

Objective: Exploring the influence of gm-csf and IL-four doses’ change to theextraction purity and number on the original generation of mice bone marrow-deriveddendritic cells and finding proper PEG molecules for fusing dendritic cells andmalignant melanoma cells.Methods: Take4weeks SPF male C57BL/6mices and broken neck put them todeath. After the separation of the mice tibial and femoral, takeing the bone marrowcells.Useing the completely culture medium to resuspend cells and culture them in6orifice plate, each hole with1.5ML completely culture medium, and then put them into37℃constant temperature incubator containing5%CO2concentration.3hours later,abandoning the supernate fluid, and then culture the cells with diffferent doses ofGM-CSF (200、500、1000) and IL-4(200、500、1000) of1640medium.5days later，joining TNF-α1.5ML to stimulate cells mature. To the cells from different groupsrespectively, we use the Inverted microscope to observe cells form、the cell count plateto count,FACS to analyz the change of expression(MHC-Ⅱ, CD11c, CD80, CD83)under different maturity、detect the purity of cells, immune fluorescence technology todetermine CD83expression of mature cells. We prepare7days mature DCs andexponential phase melanoma cells to cell suspension with uncompletely culture medium.After using PKH67-GL and PKH26-GL to stain DCs and melanoma cells respectively,utilizing PEG-3000and PEG-4000to prepare fusion cells.We observe cells form andestimate of the number of cells and purity with Leica DMIRB.Results: To the GM-CSF concentration for1000U/ml, IL-4concentration for500U/ml group of dendritic cells, cell density moderate, dendritic structure like pseudopodsobviously, presenting the typical dendritic cells structure,which obviously better thanthe other three groups (P <0.05). We use FACS to analyze the change of DCs phenotypes and demonstrate that some DCs phenotypes change when its’ cells maturitychange. DCs immune fluorescence show high expression of CD83of mature DCs.Under the Leica DMIRB we find that the group fused with PEG-3000has a greatnumber of cells stain with PKH67-GL（green）and PKH26-GL（red）,at the same time,wecan observe some fusion cells emitting yellow and bigger than green and red cells.ThePEG-3000group has a fusion rate of40%-50%which is better obviously thanPEG-4000group.