The Separation, Purification And Evaluation Of Mature Dendritic Cells From Mice Bone Marrow

Objective: Dendritic cells is the most powerful antigen presenting cells,it is so far the only one that can stimulate initial T cell activation andproliferation. It can be mediated antigen transfer, activated resting T cells inthe body, started the specific immune response. Multiple sclerosis is a chronicinflammatory disease of the central nervous system,it be mediated by T cells.Its pathogenesis has not yet had a clear,,but many researches show that MS isconnected with the abnormal activation of T cells, and,the pathologicalimmune response of MS and the participation of DC has a direct relation.Based on the characteristics of DC, in recent years, the research that used DCin autoimmune disease has been thorough development, especially itsapplication in the treatment of multiple sclerosis has become the hotspot.Therefore, to correct and efficient separate cultivate DC and carries on theappraisal is the foundation and premise of the DC downstream experimentWe have designed this experiment, aims to use the method that cultivatemature dendritic cells from mice bone marrow induced by external combiningseparation and purification with immune magnetic beads to obtain DC andfrom morphological observation, the cell surface molecular detection andstimulate the T cells appreciation ability to identify DC, explore and optimisethe method of separation,purification and appraisal of mice mature dendriticcells from bone marrow,for the further study for the application of DC inmultiple sclerosis treatment provide experimental basis.Methods:1The separation and cultivation of mature DC from mice bone marrowExcute C57BL/6mice by pull the neck, bathed in75%ethanol in3to5minutes, remove the femur sterile,prick both ends of the femoral medullarycavity with needles and unicom them, suck RPMI1640cultures with a syringe to rush out the bone marrow, filter and add the red blood cells cracking liquidto dissolve red blood cells, adjust cell concentration to106/ml by RPMI1640completely cultures(contain10%tire bovine serum, rmGM-CSFS (10ng/ml),rmIL-4(10ng/ml)), vaccinate in six holes training board, train the cellculture plate in the incubator that contains37o C,5%of of CO2,add rmTNFalpha (15ng/ml) in the fixth day and continue to cultivate to the seventh day,collect all the cells suspended, that is the mice mature DC from bone marrow.2. Purification DC with CD11c immune magnetic beads2.1Mark DC with CD11c immune magnetic beads: Count to DC,suspended it with buffer liquid. Add CD11c immune magnetic beads to it,incubate15minutes in2-8o C and suspended it with buffer liquid one moretime2.2Separated DC by magnetic beads sorting device: placed the LScolumn in the MiDi MACS sorting device. Add buffer liquid into the LScolumn wash embellish LS column. Add the magnetic beads labeled cellssuspension liquid into sorting device, collect effluents, this is not labeled cells.Move up column from sorting device,add5ml buffer liquid, push the markcells out with the matching pistons.3. Identification of the mice mature dendritic cells from bone marrow3.1Cell morphology observation3.11Observe the morphologyofDC with convert fluorescence microscopy3.12Observed DC form with scanning electron microscopy (sem) andtake photos3.13Observed DC form with transmission electron microscope and takephotos3.2The detection of cell surface molecules by flow cytometric: Collectsuspended cell in the culture plate, after the centrifugal, washing, counting, setthe capacity and mark, add the antibody to the cells, incubate avoid light inroom temperature20minutes and centrifugal, washing, detect the molecularexpression of CD11c, CD80, CD86, MHCII in DC surface.3.3Detect the ability of proliferation that DC stimulate the linebreed gene T in one-way mixed lymphocyte responses (one-way MLR)with themethod of cell proliferation detection reagents (CCK-8):(1) Take mature DCfrom C57BL/6mice bone marrow as the stimulation cells.(2) Take T cellsfrom BALB/c mice spleen as the reaction cells, and detect a few T cells byflow cytometric before detection (3)Purify T cells with CD90magnetic beads.(4) Detect the purity of T cells by flow cytometric.(5) DC and T cells weretraining together:the percentage of DC and T cells were set to1：5,1:10,1:20,1:40, and training72hours, before the end of the training in four hours to addCCK-8, each hole20μ L, continue to cultivate4hours and to inspecte andrecord the absorbency value in each hour after add CCK-8by enzyme leaguedetector.Results:1The morphology of mDC observation1.1Observe the morphology of DC with convert fluorescencemicroscopy: Bone marrow cells in mice by gm-csf, rmIL-4induce and aftertraining48hours, most cell on the growth visible, the size and form of cellswere different, training after72hours cells suspended increased, some cellsform colony. The sixth day suspended cells continue to increase, and appear afew of dendritic sample bumps. Add rmTNF alpha into the cell and training24hours, cell surface appear typical branched bumps, accord with mature DCform.1.2Observe the morphologyof DC with scanning electron microscopy：the form of DC is various, and the structure is intact, the surface is rough, isseems to be covered with a layer of gauze,there are large branched bumps anddrape, accord with mature DC form.1.3Observe the morphology of DC with transmission electronmicroscope: DC body is large, cell shape is irregular, the cell surface has along dentrites, different length, uneven thickness., the surface has branchedbumps;nuclear membrane is clear, the organelles in cytoplasm are rich,obviously, a lot of golgi body, endoplasmic reticulum, mitochondria, andribosomes, but saclike structure and the lysosome reduced. 2. The detection of cell surface molecules by flow cytometric:In maturedendritic cells from mice bone marrow,CD11c is highly expressed, purity ismore than90%. The cell surface CD80(94.73%±1.45%), CD86(96.43%±2.00%), MHCII (94.13%±5.86%), are highly expressed, accord with matureDC's express features.3. The proliferation of ability that DC stimulate linebreed gene T cell:After the separation of magnetic beads,T cells's purity is more than90%bystreaming testing,each stimulus index of the proportion of DC: T cells canexplain that DC have the ability of proliferation which exciting linebreed geneT cells,but in2,3hours that after add CCK-8into cells,test the stimulationindex in different proportion of DC: T cells respectively,there was nostatistical difference; the proliferation of ability that DC stimulate T cell inproportion to the proportion of DC in the forth hour. The differences betweeneach group have statistical significance.(P0.05)Conclusion:1This experiment use the method that induce training by gm-csf,IL-4and TNF-αin vitro combine with purify DC by immune magnetic beads,Successful culture and the augment high purity mature DC, mature DCdeveloped using this method has typically dendritic form, quantity is big, highpurity.2Through three aspects cells appraisal:the morphological observation,the detection of cell surface molecules by flow cytometric and one-way mixedlymphocyte reaction, proved this DC is mature DC. In the determination ofproliferous ability that DC stimulate T cells by mixed lymphocyte responsesof one-way, we use CCK-8method has replaced traditional MTT method, andmakes the experimental results more stable, reliable, and the experimentalresults also show that best measured time use CCK-8is add CCK-84hourslater,and the proliferation of ability that DC stimulate T cell in proportion tothe proportion of DC in the forth hour.3This experiments discussed separation,training in vitro, purification andappraisal of mature dendritic cells from mice bone marrow,and for the study of DC and its application in treatment of multiple sclerosis to provideexperimental basis.