| Objective:To observe the effect of bone marrow-derived Dendritic cells(DC) toxicity under different concentrations of atmospheric PM2.5 in BALB/c mice.Methods:1.Atmospheric PM2.5 collection: From January 1st,2014 to February 1st,2014,collected the PM2.5 filters in relatively pollution areas and relatively clean areas of Taiyuan City in Shanxi province. PM2.5 would be made into freeze-dried powder. Before using it,ultraviolet lamp irradiate the freeze-dried powder for 30 minutes, to dissolve PM2.5 freeze-dried powder with cell culture fluid containing 10% fetal bovine serum RPMl l640,and make particle suspensions of different concentration, concentration of PM2.5 included with 50ug/ml,100ug/ml,200ug/ml,400ug/ml and 800ug/ml.2.Culture the mouse dendritic cells(DCs) in vitro: to extract BALB/c mice bone marrow cells under sterile conditions and separate the mononuclear cells.The cells were cultured with recombinant granulocyte macrophage colony stimulating factor(20ng/ml GM-CSF) and interleukin(IL-4) 2ng/ml synergistically for a week. To observe the morphological changes of DC under inverted microscope, and to observe the phenotypic characteristics of DC with immunofluorescence staining.3.Inoculate the activitive dendritic cells in 12 well plates, the number of cells in each hole was 1 x 104. Cells were divided into 6 groups, the control group with RPMl containing 10% fetal bovine serum l640 cell culture fluid, and the other 5 groups with PM2.5 of different concentrations containing 10% fetal bovine serum RPMl l640.The 6groups were cultured for 24 hours.After 24 hours,collect the cells.To observe the survival rate of DC cells with the MTS method.4.Analysising the activity of lactate dehydrogenase(LDH) in the cell culture fluid,to judge the damage degree of PM2.5 to DCs.5.Inoculate the activitive dendritic cells in 12 well plates, the number of cells in each hole was 1 x 104. Cells were divided into 4 groups randomly :the control group,deferoxamine mesylate group, DFO+ PM2.5 group and PM2.5 group. Dendritic cells were cultured for 24 hours, use the MTS method to observe the survival rate of DC cells,to judge whether the PM2.5 mixture composition containing metal ion?6.Use the DAPI/PI to dye the DC and observe the ratio of apoptosis / death of DC under a fluorescence microscope.Results:1.Obtain bone marrow mononuclear cells and activitive DCs successfully.2.The effects of PM2.5 of different concentration to the rate of survival of DC:the survival rate of cells was 99.2% in the control group, the survival rate of other groups which the concentration of PM2.5 was 50 g/ml, 100 g/ml, 200 g/ml, 400 g/ml, 800 g/ml were 88.10%, 81.45%, 71.48%, 43.65%, 21.82%.3.Analysis activity of LDH :The concentration of PM2.5 was 50 g/ml, 100 g/ml,200 g/ml, 400 g/ml, 800 g/ml.The of LDH were 5.5%, 14.8%, 27%, 78%, 89% in each group. The amount of LDH and PM2.5 concentration showed a dose dependent relationship.4.Control group, DFO group, DFO+ PM2.5 group, PM2.5 group, the survival rate of cells was 100% in the control group,the survival rate of other groups :95.41%, 75.85%,22.38%.5.Use the DAPI/PI to dye the DC, observe living cell survival rate, apoptosis rate,mortality rate under fluorescence microscope : between the PM2.5 group and Control group, DFO group, DFO+ PM2.5 group had significant difference.Conclusions:The toxic effect of PM2.5 on bone marrow derived dendritic cells is exist,with the dose increasing, the toxicity increase as well.The component of PM2.5 is complex, toxic effects on bone marrow derived dendritic cells come from not only soluble in aqueous phase material, metal ion also plays an important role. |
PDF Full Text Request