In Vitro Research On The Effects Of Mobilization Reagent DMOG On MSCs Biological Characteristics And HIF-1 Signaling Pathways

Objective Our project group has foud that prolyl hydroxylase small molecular inhibitor DMOG could mobilize MSCs,and in vivo experiments showed HIF-1 signaling pathway played an important role in mediating MSCs mobilization with DMOG On the basis of our early results,in this study,we investigated the influence of different concentration of mobilization reagent DMOG on MSCs biological characteristics and its regulated effect to HIF-1 signaling pathways by in vitro researches.Methods(1)In vitro research on the effect of different concentration of mobilization reagent DMOG on rat MSCs biological characteristics:?The whole bone marrow adherent method was used to culture the SD rat MSCs,and the experiment was divided into DMOG O?M group,DMOG 2 0?M group,DMOG 40?M group,DMOG 80?M group;? The effect of DMOG on MSCs proliferation ability was detect by CCK-8 assay;?We used flow cytometry to analyse the influence of DMOG on MSCs cell cycle;?Transwell method was used to explore the effect of DMOG on MSCs migration ability;?The effects of DMOG on MSCs osteogenic,adipogenic,chondrogenic and neural differentiation abilities were investigated with the method of induction differentiation solution incubating.(2)In vitro research on the regulated effect of different concentration of mobilization reagent DMOG on HIF-1 signaling pathways in rat MSCs:?After HIF-1? siRNA being built,it was transfected into rat MSCs with the method of RNA interference;the optimum dosage of siRNA was filtered by detecting transfection rate with positive control;Then,the experiment was divided into Control group,transfected Control siRNA group,transfected HIF-la siRNA group;?Western blot assay was used to detect the influence of DMOG treatment on HIF-1?,SDF-1?,VEGF protein expression before and after HIF-1? being interfered;?Real-time qPCR method was adopted to examine the effect of DMOG treatment on HIF-1?,SDF-1?,VEGF mRNA expression before and after HIF-1? being interfered.Results(1)In vitro research on the effect of different concentration of mobilization reagent DMOG on rat MSCs biological characteristics:?MSCs cultured by whole bone marrow adherent method were pure with high homogeneity and strong cell vitality that could be used for following study.?The result of CCK-8 showed that the cell growth curve of every group was close to S type after different concentrations of DMOG treatment.DMOG 20?M processing began to promoted cell proliferation ability of MSCs(P<0.05),and the promotion effect of DMOG 40?M was the most obvious(P<0.05).?Flow cytometry result showed that cells proportion of S phase+G2/M phase in MSCs was improved with DMOG treatment,and there were no significant differences between three concentrations(P>0.05).?Transwell result showed that DMOG treatment could promote the migration ability of MSCs,and the promotion effect was statistically significant of DMOG 20?M processing(P<0.05).?The results of induced differentiation showed that after processing with different concentration of DMOG,MSCs still kept the original osteogenic,adipogenic,chondrogenic,neural differentiation abilities.DMOG 20?M treatment could obviously promote osteogenic and chondrogenic differentiation abilities(P<0.05),suppress adipogenic differentiation ability,had no significant effect on neural differentiation ability of MSCs(P>0.05).(2)In vitro research on the regulated effect of different concentration of mobilization reagent DMOG on HIF-1 signaling pathways in rat MSCs:?Through transfection rate detection with positive control siRNA kits,the optimum dosage of siRNA was 30pM,and the final concentration was 10nM.?The result of Western blot showed that on one hand,before HIF-1? siRNA interference,DMOG 20?M treatment could obviously upregulate protein expression of HIF-la and its downstream SDF-1?,VEGF in MSCs;On the other hand,HIF-1? siRNA interference could effectively reduce HIF-1? protein expression,and its downstream SDF-1?,VEGF protein expression decreased after that(P<0.01),at the same time,the protein expression level of HIF-1??SDF-1??VEGF could be upregulated with DMOG 20?M treatment(P<0.01).?Real-time qPCR result showed that on one hand,before HIF-la siRNA interference,DMOG 20?M treatment could obviously upregulate the mRNA expression level of HIF-la and its downstream SDF-1?,VEGF in MSCs;On the other hand,HIF-la siRNA interference could effectively reduce HIF-1? mRNA expression,and its downstream SDF-1?,VEGF mRNA expression decreased after that(P<0.01),at the same time,the mRNA expression level of HIF-1??SDF-1??VEGF could be upregulated with DMOG 20?M treatment(P<0.01).Conclusion(1)After processing with different concentration of DMOG,MSCs still kept the original multipotent differentiation abilities.20?M DMOG treatment could obviously promote osteogenic and chondrogenic differentiation abilities,suppress adipogenic differentiation ability,have no significant promoting or inhibiting effect on neural differentiation ability of MSCs,at the same time could improve the proliferation and migration abilities of MSCs.(2)Before or after HIF-la being interference,DMOG 20?M treatment could obviously upregulate the protein and mRNA expression level of HIF-1?and its downstream SDF-1??VEGF.It remindered the regulated effect of DMOG on HIF-1 signaling pathways in MSCs.And it further confirmed that HIF-1 signaling pathways played an important role in DMOG inducing MSCs mobilization from the perspective of in vitro experiments.