| Objective:To compare the form and function between C57BL/6 mice-derived primary myeloid dendritic cells(DC) and DC2.4 genetically modified.To compare the difference between different dose of erigeron extract on DC2.4' maturity and function.Methods:Collect C57BL/6 mice bone marrow cells in long bones,through semiadherent DC precursors was obtained and cultured with granulocyte - macrophage colony-stimulating factor(GM-CSF) and interleukin-4(IL-4),then induced with lipopolysaccharide(LPS) to stimulate DC2.4 and DC after 7d for 48h.observated morphology and detectted IL-12 concentration and surface marker CD1 1c,CD80,CD86 and MHC-Ⅱ;DC2.4 was divided into negative(simple culture fluid),positive(induced by LPS) and experimental group(add different dose after LPS induction),experimental group was divided into four groups from 1 to 4,respectively adding different dose of extract(50,100,150和200μg/ml) for 3d,then compare morphology,detect IL-12 and surface markers CD1 1c,CD40 and MHC-Ⅱ,through mixed lymphocyte culture to detect the effect of stimulating T cell to proliferate.results:Comparison between DC from mouse marrow cells and DC2.4: morphological observation under inverted microscope on 3d monocytes adherent macrophages and semi-adherent cells can be seen,the size become large,irregularly shaped or tadpole-shaped or spindle,with a large number of processes for DC;on 4～7d shows that some cells increased and get off the wall,showing the characteristic star-shaped,with 2 to 5 processes and some longer,but still spindle;after adding LPS 48h on 7d,lots of DC get off the wall with more branches and coarse.DC2.4 was semi-adherent,star-shaped,tadpole-shaped or spindle-shaped,it was similar with the mouse bone marrow-derived dendritic cells.After adding LPS dendritic branches increased and thickened.DC group by LPS-stimulating the concentration of IL-12 was(32.01±2.62) pg/ml,DC group cultured for 9d(14.36±1.98)pg/ml,the difference was significant(P＜0.01);LPS-induced DC2.4 group concentration(29.92±5.76)pg/ml, and DC2.4 group(14.98±4.75)pg/ml and the difference was significant(P＜0.05). FACS analysis DC2.4 in CD1 1c,CD80,CD86 and MHC-Ⅱexpression rate (1.62±0.51)%,(2.03±0.15)%,(8.19±1.13)%,(13.88±12.62)%,LPS after induction(5.54±1.08)%,(2.42±0.20)%,(9.59±1.27)%,(31.45±12.09)%,in CD1 1c and MHC-Ⅱdifference was significant(P＜0.05);9d DC culture and to 48 h after LPS stimulation,DC of CD1 1c,CD80,CD86 and MHC-Ⅱrate(55.11±4.48)%, (40.68±3.77)%,(29.91±3.59)%,(41.27±2.95)%and(84.25±4.62)%,(74.61±4.94)%,(61.50±5.10)%,(57.41±3.31)%,there were difference in every marker (P＜0.01).DC2.4 after dealing with erigeron extract under inverted microscope,DC2.4 was semi-adherent,astro-shaped,tadpole shaped or fusiform shaped,the same with DC. After adding LPS,branches increased,thickening,round,gradually enlarged, polygon-shaped and distinctively astro-shaped,caused by membrance and ketoplasm.after adding extract cell become na(i|¨)ve,rare,the more obvious the high dose. Under TEM DC2.4'surface projection increased,cell nucleus visible and nucleoli obvious,lots of phagocytic vesicle and lipid droplet with uniform text in tracytoplasm.Under SEM DC2.4 after LPS branches and projection increased.In positive group level of IL-12 for 29.92±5.76pg/ml,compared with the negative group and experimental group,there were statistically significant differences (P＜0.01).In addition to the minimum and highest dose group P＜0.05,the difference was not obvious between other group P＞0.05.DC2.4 markers In each erigeron group CD11c in addition to the experimental group 2 and 3 with no significant difference,the expression rate from high to low,the significant difference between groups(P＜0.05 or P＜0.01);in CD40 experimental grouplhigher than other experimental groups(P＜0.01),there was no significant difference between group 2,3,4;in MHC-Ⅱexperimental group 1 higher than group 4(P＜0.05),there were no significant difference between other experimental groups(P＞0.05).Conclusion:1.DC2.4 owns the majority of properties with C57BL/c-derived dendritic cells and can be used as an alternative in vitro model because of high purity, time -saving and low training costs.2.LPS has the promotion of maturity of primary cells and DC2.4.In morphology,surface markers and secretion of cytokines,the DC and DC2.4 of the stimulus is not exactly the same with each other.3.The state of DC2.4 and function intimatly correlate with morphology,surface markers and cytokine.4.Erigeron extract can inhibit DC2.4' maturity,along with dose increasing, the inhibitory action gradually reinforce.5.DC2.4' function correlates with its mature state,with its maturity,the effect of presentting antigen gradually reinforces.
|
PDF Full Text Request