The Influence On Expression Of Smad3、Smad4、RUNX2 In BMSCs With ZHX3 Gene Silencing

PurposeTo investigate the influence on expressions of smad3、smad4 and RUNX2 in bone marrow mesenchymal stem cells(BMSCs)induced by zinc fingers and homeoboxes 3 gene(ZHX3)silence.MethodsCells were divided into three groups including ZHX3 silent group(ZHX3interference vector was constructed and it’s recombination virus particles were packaged,then the particles were transfected into the rat BMSCs),vehicle control group(Empty vector was transfected into BMSCs)and control group(Wild type BMSCs).Cell transfection rate was measured under a fluorescence microscope,then identified whether transfection is successful.Osteogenic ability of cells was detected by alkaline phosphatase staining after osteogenic induction.Using immuhistochemistry and western blotting methods detect the expression level of smad3、smad4、RUNX2 after ZHX3 silence.Results(1)After transfection,ZHX3 silent group and vehicle control groups were expressing green fluorescent,blank control group did not express fluorescence and expression level of ZHX3 in ZHX3 silent group was significantly lower than the control group and vehicle control group.(2)cells in each group show alkaline phosphatase positive expression cells and mineralized nodules,however,the quantity of the latter are less than the former two.(3)Under a fluorescence microscope Observed that expression of smad3,smad4 located in the nucleus and cytoplasm,expression of RUNX2,mainlylocalized in the nucleus.Positive cells were seen In three groups,and the fluorescence intensity between the control group and the vehicle control group was no significant difference,but the fluorescence intensity of ZHX3 gene silencing group was significantly lower than the two control groups.(4)Western blotting analysis showed that expression of smad3,smad4,RUNX2,was no significant difference in the control group and the vehicle control group,but were significantly higher than ZHX3 silent group.ConclusionZHX3 gene silence can delay the osteogenic ability of BMSCs in vitro,which may through the down-regulated expression levels of smad3,smad4,RUNX2.