Roles Of Jmjd3 In Site-specific Of Bone Marrow Stromal Cells Senescence

Objective:To investigate the effects of histone demethylase Jumonji D3(Jmjd3)on the senescence of rat bone marrow stromal cells(BMSCs)from different embryonic origins.Methods:1.BMSCs were extracted from mandibular(M)and tibia(T)of rats in vitro,and were cultured and passaged.2.BMSCs were collected,centrifuged,and seeded to the cell culture plate at approximately 5×10~4 cells per well.When the cell fusion reached 70%-80%,senescence-related?-galactosidase(SA-?-gal)staining was performed.Acquiring images of aging cells were counted under a microscope to detect the cell senescent characteristics.3.BMSCs were collected,centrifuged,and seeded into a 12-well cell culture plate at approximately 2×10~4 cells per well.When the cell fusion reached 60%-70%,the osteogenic induction solution was replaced,and the medium was changed every three days.After 14 days of continuous osteogenic induction culture,alizarin red staining was performed to detect cell osteogenesis characteristics by calcium nodule formation ability.4.The expression of the target genes Jmjd3,p21,p16 were detected in M and T group by quantitative Real-time PCR.5.Relative expressions of Jmjd3,p21 and p16 proteins in BMSCs were detected by Western Blot.6.Then,shJmjd3 Lentivirus was constructed and transfected into T-BMSCs to knockdown Jmjd3 expression,and the characteristics of cell senescence were detected by SA-?-gal staining,and the expression of Jmjd3 and senescence related factors,p16 and p21,were detected in each group by Western Blot.7.Rats were divided into 5 groups and each group has 6 rats.Then,a critical-sized bone defect model was established in the rat mandibular region,and a mixture of stent and cell formation was transplanted into the defect area.The bone-healing region after BMSCs transplantation was detected by Micro-CT and Masson staining.Results:The results of SA-?-gal staining showed that the number of senescent cells in T-BMSCs group was significantly higher than that in M-BMSCs group(P<0.05),and the ability of osteogenic differentiation of M-BMSCs was stronger than that of T-BMSCs(P<0.05),the difference between the two sets of results was statistically significant.Quantitative Real-time PCR and Western Blot showed that Jmjd3,p21,p16 genes and proteins were highly expressed in T-BMSCs group and the difference was statistically significant(P<0.05).In SA-?-gal staining,senescent cells in T-BMSCs group transfected by shJmjd3 virus were significantly decreased than the control group.Jmjd3,p21,and p16 of T-BMSCs transfected with shJmjd3 virus were showed low expression by Western Blot.In cell transplantation experiments in vivo,the T-BMSCs group with shJmjd3 transfection showed that the bone mean density and bone volume fraction were significantly increased by micro-CT.Compared with GFP T-BMSCs,rats transplanted with shJmjd3 T-BMSCs exhibited the elevated trabecular bone structure by Masson staining.Conclusions:M-BMSCs exhibited lower senescence and higher osteogenesis characteristics than that of T-BMSCs,which have a higher Jmjd3 expression.Inhibition of Jmjd3 in BMSCs can enhance the anti-senescence ability of cells and promote bone repair in vivo.