| Objective:The biological basis of orthodontic tooth movement is bone remodeling,and there are many factors affecting alveolar bone remodeling.Mechanical force is one of the most important factors.Bone marrow mesenchymal stem cells are proliferated and differentiated by mechanical stimulation,which is important for orthodontic tooth movement and orthodontic maintenance.This experiment begins with the molecular level of cells,and further explores the signaling pathways and mechanisms of stress-induced proliferation and differentiation of rat bone marrow mesenchymal stem cells,and provides a theoretical basis for clinical treatment of more scientific and rational orthodontic treatment.Methods:1.Rat bone marrow mesenchymal stem cells were obtained by whole bone marrow adherence method.After purification,the cells were observed for morphology,the growth curve was drawn by cell counting,and the cell surface antigen was identified by flow cytometry.2.The in vitro culture-tension stress loading model of rat BMMSCs was constructed.The loading stress of BMMSCs was 1 Hz,and the tensile tensile deformation was 5%.The tensile stress lasted for 0h,3h,6h,9h and 12 h.Western blot and q RT-PCR were used to detect the protein and m RNA expressions of osteogenesis-related factors RUNX2,BMP2 and transcription factors Twist,E2 A and p21 in different stress loading time.3.To design and synthesize four sh RNA oligo sequences and a negative control sequence for rat p21 gene sequence,insert into lentiviral vector to construct lentiviral expression plasmid,and transform into competent cells for sequencing.The concentrated virus stock was collected after lentivirus packaging and the virus titer was determined.Rat BMMSCs were infected with four groups of p21-sh RNA lentivirus and negative control lentivirus at a multiplicity of infection of MOI=100,and the interference effect of lentiviral vector on p21 gene was detected by q RT-PCR.The lentivirus with the best silencing effect and the lentivirus with negative control were selected to infect BMMSCs to construct stable strains.4.Set blank cell group(WT),p21 gene silencing group(p21-shRNA),negative control group(NC),flow cell analysis to detect cell cycle,q RT-PCR analysis without silencing,silencing p21 to transcription factor Twist,The effect of E2 A and the transcription of the relevant factors RUNX2,BMP2,Osterix.The cyclic tensile stress of 1 Hz,5% and 9h was applied to the three groups of cells.Western blot and q RT-PCR were used to detect the osteogenesis-related factors RUNX2,BMP,Osterix and transcription factors of rat BMMSCs with p21 after cyclic stress.Changes in protein and m RNA expression of Twist and E2 A,and exploring the mechanism of mechanical stimulation mediated osteogenic differentiation of rat BMMSCs.Results:1.Rat bone marrow adherence method can obtain rat BMMSCs.After in vitro adherent culture and subculture,the cell growth is gradually stable,and the cell morphology is uniform and long fusiform.The BMMSCs passed to the 3rd to 5th generations had a uniform density distribution and showed an order of fibroblast-like morphology.The growth cycles of P1,P3 and P7 cells were divided into incubation period,logarithmic growth phase and growth plateau,but the cell proliferation rate of early passage(P1,P3 generation)was always higher than that of late passage(P7 generation).The results of flow cytometry showed that the cell surface antigens CD44,CD90 and CD105 were positive,while the CD4 and CD45 results were negative.The above results were consistent with the surface specific antigen of rat bone marrow mesenchymal stem cells.2.After loading 1Hz,5% periodic tensile stress,BMP2(**P<0.01)and Runx2(**P<0.01)all showed a time-dependent increase with the increase of the afterburning time.Transcription factor TWIST(*P<0.05)and p21(**P<0.01)also showed a timedependent increase,while m RNA and protein expression of transcription factor E2A(***P<0.001)decreased with time.It is indicated that cyclic tensile stress can promote osteogenic differentiation of rat BMMSCs and affect the expression of transcription factors.3.After alignment,the fragment sequence inserted in the recombinant lentivirus was identical to the designed oligo sequence,and the p21 RNA interference lentiviral vector was successfully constructed.The virus titer was 5×108 TU/ml,and the efficiency of transfecting BMMSCs was greater than 80%.4.Compared with the WT group and the NC group,the proportion of S phase cells in the p21-sh RNA group was significantly increased,and the cells in the G1+S phase were also increased,and the proportion of cells in the G1 and G2 phases was decreased.Silencing the p21 gene of BMMSCs caused a small decrease in the relative expression of transcription factor Twist m RNA(*P<0.05),a small increase in E2 A m RNA(*P<0.05),osteogenesis-related factor BMP2(*P<0.05),Runx2 The relative expression levels of m RNA(**P<0.01)and Osterix(**P<0.01)were lower in the p21-sh RNA group than in the WT and NC groups.5.After loading the cells of WT group,p21-sh RNA group and NC group with periodic tensile stress of 1 Hz,5% and 9 h,the results of q RT-PCR showed that after silencing p21,the transcription factor Twist(**P<0.01)m RNA expression was inhibited,and m RNA expression of E2A(***P<0.001)was greatly promoted;m RNA expression of osteogenic factors Runx2(**P<0.01)and Osterix(***P<0.001)was detected in p21There was an increasing trend after inhibition,and BMP2(*P<0.05)showed a decreasing trend with the silent table of p21.The protein results for the above factors are consistent with the trend of q RT-PCR.Conclusion:1.Periodic tensile stress promotes osteogenic differentiation of rat bone marrow mesenchymal stem cells in vitro and affects the expression of transcription factor Twist/E2A/p21.2.p21 plays an important role in maintaining the relative tension induced by BMP2 and Runx2-Osterix.3.During cyclic osteogenic induction of osteogenic differentiation of rat BMMSCs,Twist,E2 A and p21 are not simply linearly regulated,and p21 negative feedback regulates the expression of Twist and E2 A. |
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