The Separation And Evaluation Of Dendritic Cells From Mice Bone Marrow And Preliminary Evaluation Of The Total Flavonoids On Gout

Objective 1.The dendritic cells were isolated from bone marrow of mice by adding to cytokine GM-CSF and IL-4,then used in vitro research.2.The purity of dendritic cells was identified by morphological observation and molecular detection of cell surface,which provided experimental basis for further study.3.Research the flavonoids on Smilax on the key factors of gout IL-1?,TNF-? and related protein caspase-1,preliminary evaluation effect of flavonoids on Smilax on gout,which provide the basis for further study on the mechanism of TFSG on gout.Methods 1.Cells Culture:Mice were sacrificed by cervical dislocation,aseptic femurs,the bone marrow cells were washed out.Added 1×106 cells/well to 6-well plates in RPMI 1640 medium,added FBS,GM-CSF and rmIL-4,and differentiated at 7 days,then obtained BMDCs.2.Identification of the mice dendritic cells from bone marrow:2.1 Identification of the mice mature dendritic cells from bone marrow.2.2 The detection of CD86 cell surface molecules by flow cytometric:Collected suspended cell in the culture plate,after the centrifugal,washing,counting,seted the capacity and mark,added the antibody to the cells,incubated in dark at room temperature for 20 minutes and centrifugal,washing,then detected the molecular expression of CD86 in DC' surface.3.The detection the safe concentration range of TFSG:observed different concentrations of TFSG on the growth of DCs by MTT method.First,about 5x103/hole cells were seeded in 96 well plates,then added different concentrations of TFSG,these cells were incubated at 24 hour(h),48h,72h.At each time,MTT solution was added and then incubated for 3h(purple crystal formation),then added DMSO solution,finally readed(570nm wavelength)the OD in the microplate.4.The effect of flavonoids on Smilax:The BMDCs(1×106 cells/ml)were divided into six groups,namely normal group,model group,positive group,Smilax total flavonoids group(low,medium and high dose groups).Then followed by sodium urate(MSU)crystals(100?g/ml)and mixed with a certain volume of Smilax total flavonoids(100,300,500?g/ml)or positive control drug colchicine(0.1 ?g/ml),then incubated for 20h at 37?.Normal group,model group replace drugs with culture medium,the test was repeated three times.Finally,the IL-1?,TNF-? on the supernatant was measured by ELISA method and caspase-1 expression level was detected by Western blot.Results 1.Observed the morphology of DCs with convert fluorescence microscopy:Bone marrow cells in mice was discarded supernatant after training 24 hours,then by GM-CSF,rmIL-4 induce 48h and most cells on the growth visible,the size and form of cells were different,training after 72 hours cells suspended increased,some cells formed colony.The suspended cells continued increasing at the sixth day,and appeared a few of dendritic sample bumps.Added LPS into the cell and continued training for 24 hours,cells surface appeared typical branched bumps,accorded with DCs form.2.The detection of cell surface molecules by flow cytometric:the expression of the cell surface CD86 was ranged from 9.5%to 32.5%,which was in accordance with DC's features.3.By ELISA method,the Model group of the IL-1?,TNF-? level higher than normal group,higher concentration of TFSG reduced the level of IL-1?,TNF-?;by Western blot,higher concentration of TFSG decreased the expression level of Caspase-1.Conclusion 1.This experiment used the method that derived and cultivated BMDCs by adding GM-CSF?IL-4 and LPS into BMDCs.2.The higher concentration of TFSG had a certain effect on the main factor IL-1?,TNF-? in gout,and the expression level of relative protein caspase-1 was down regulated,which could be a preliminary evaluation of the therapeutic effect of TFSG on gout.