The Effect Of Deoxynivalenol On The RUNX2Experssion In Mouse Osteoblast

ObjectiveDiscuss two methods of osteoblast primary culture. To detect the effect of DON at thedifferent concentration on mouse osteoblast cell cycle and apoptosis; to detect theexpression changes of Runx2. Provide a theoretical basis for the further study ofmycotoxin cause bone deformities.MethodsUsing tissue culture and collagenase II stage digestion, obtain primary cultured cellsfrom suckling mouse parietal bone and skull. Alk staining to identify the primary culturedosteoblasts. Osteoblast cells were cultured in different concentrations of DON. After PIstaining, using flow cytometry to detect the cell cycle of osteoblast; After PI-AV staining,using flow cytometry to detect the apoptosis of osteoblast; Using real time PCR to detectthe expression trend of Runx2on the influence of different concentrations of DON.Results1. Primary culture and identification: Both primary methods can obtain the mouseosteoblast cells. Tissue culture method is slowly, obtain less, but high purity. CollagenaseII stage digestion is fast, obtains more, but low purity and need purification. Alizarin redstaining and alkaline phosphatase staining results were positive, confirm that culture cell isthe osteoblasts.2. The effect on cell cycle: cell proliferation decreased with increasing DONconcentration.100ng/ml,200ng/ml group compared with control group cell proliferationwere decreased not significantly.500ng/ml group compared with control group cellproliferation were decreased significantly, the S-phase cells began to increase, and theG2/M phase cells started to decrease.1000ng/ml group acting on cells, the S-phase cellsincreased significantly, and the G2/M phase cells decreased significantly. In summary, DON affects the cell cycle. The main function of DON is S-phase arrest; obviousanti-proliferative.3. The effect on induce apoptosis: According to the results of flow cytometry: Usingdifferent concentrations of toxin to cultured mouse osteoblasts24h, with the increase in theconcentration of DON, showing the trend of increased apoptosis.100ng/ml、200ng/mlgroup compared with control group apoptosis were increased not significantly.500ng/mlgroups compared with control group apoptosis began to increase.800ng/ml、1000ng/mlgroup acting on cells, apoptosis increased significantly. It can be seen, DON can induceapoptosis in mouse osteoblasts.4. The effect on Gene expression: the same brightness of the internal reference bandson the gel between200-300bp. Gradually reduced brightness of the target bands on the gelbetween300-400bp. Under the conditions of internal reference bands brightness consistent,the brightness of the target band decreased with the increasing DON concentration. Insummary, with the increased concentration of DON, mRNA of Runx2content decreased.5. Real-time PCR results: each group internal reference Ct is consistent. Runx2geneexpression levels in each experimental group Runx2gene expression levels in eachexperimental group as follow：100ng/ml group is about72.8%of the control group,500ng/ml group is about60.9%,1000ng/ml group is about37%. Each experimentalgroup Runx2gene expression differences were statistically significant.Conclusion1. DON has a direct effect on mouse osteoblasts and inhibits osteoblasts growth,making cell S arrest, blocking cell to enter G2/M phase.2. DON can significantly induce apoptosis significantly and anti-proliferative effect.3. In the different concentrations of DON, Runx2gene expression in the osteoblastswas reduced gradually and significantly with the increasing concentration of DON.