Sduty On Influence Of Structure And Function To Dendritic Cells About Antibiotic Induced Small Intestinal Dysbacteria Mice

Objective:We analyzed these changes of shape, number, subtype, function about DCs byanalyzing between dysbacteria mice model and normal control mice.We can exploreinfluence of dysbacteria in the structure and function of dendritic cells in the smallintestine.Method1. Analysis of flora in small intestine about antibiotic induced dysbacteria micemodel: the experiment was divided into two groups, normal control group anddysbacteria group.Normal control groups was fed with sterile water, dysbacteriagroups was fed with the400mg/ml ceftriaxone sodium. The sterile water orceftriaxone sodium was0.2ml each,2times a day, the interval of time was6h, thesemices were fed with8d; finally we analyzed small intestinal flora of these mices, andestablish dysbacteria mice model.2. Separation,purification of small intestinal dendritic cells in mice,(1)Prepartion single cell suspension by method that EDTA shock to remove theepithelial cells and type IV collagenase digest small intestinal tissue.(2)Separation and purification of the small intestinal dendritic cells in mice byCD11c+immunomagnetic beads.3. The impact of dysbacteria to small intestinal dendritic cells.(1)Observe the morphological changes of dendritic cells in small intestinelamina propria by transmission electron microscopy.(2)Detect the changes of CD11c,CD8a,CD11b,MHC-II,CD86,CD80,CD40,CD83,CD205in small intestinal dendritic cell by flow cytometry. (3)Detect the changes of IL-10, IL-12in small intestinal dendritic cells byRT-PCR.Results:1. Analysis of flora in small intestine about antibiotic induced dysbacteria micemodel: we can establish dysbacteria mice model by utilize ceftriaxone sodium.In thesmall intestine after flora imbalance,quantities and types of bacteria were significantlyreduced. Number of Bacillus bifidus, Bacteroides, Fusobacterium, Veillonella andStaphylococcus aureus significantly reduced.2. Separation and purification of small intestinal dendritic cells in mice: Someprotrusions of different sizes were observed by optical microscope around intestinaldendritic cells. But the dendrites are not obvious,due to fixation ofparaformaldehyde.After collagenase digestion and immunoma-gnetic bead, the purityof dendritic cells can reach55%-60%after two MS column, and purity becomehigher if we use multi-columns.3.Influence of dysbacteria to intestinal dendritic cells(1)Morphology of intestinal dendritic cells:Lamina propria dendritic cells ofnormal control group had the pseudopodia of varying sizes.The edge of the cell wasclear. Nucleus was irregular. Compared with the normal control group, in dysbacteriagroup, pseudopodia of dendritic cells become shorter, the edge of the cell was fuzzy,irregular.Cytoplasmn became less.(2)The number and subtypes of intestinal dendritic cells: Compared with thenormal control group, the number of CD11c+dendritic cells was significantlyincreased in the small intestinal mucosa in dysbacteria group(P=0.0001).Twosubtypes of CD11c+dendritic cells did not significantly change:CD11c+CD11b+(P=0.773), CD11c+CD8a+(P=0.235)(3)Function of intestinal dendritic cells: Compared with the normal controlgroup, in dysbacteria group, expresstion of CD205on dendritic cells was notsignificant(P=0.816).Expresstion of cell suface marker (MHC-II, CD86,CD80,CD40)was significantly reduced on dendritic cells. Its P were P=0.0001,P=0.030, P=0.036,P=0.008.Expresstion of CD83was significantly reduced(P=0.003) on dendritic cells.Expression of IL-10was significantly increased.Expression of IL-12was significantlyreduced on dendritic cells.Conclusion:1. Ceftriaxone sodium interfere flora in the small intestine, it can cause dysbacteria in mice small intestine.2. The dendritic cells were separated and purified by MACS system in the smallintestinal mucosa.3. In dysbacteria group, morphology of intestinal dendritic cells in mice waschanged, the number was increased, antigen-presenting function and maturity wasreduced. But subtypes and the phagocytosis of dendritic cell was no significant.4. In dysbacteria group, the expression of IL-10was significantly increased, theexpression of IL-12was significantly reduced on dendritic cells.