Effect Of Unidirectional Fluid Shear Force Combined With PDK1 Promoted The Differentiation Of MC3T3-E1 Cells Into Osteoblasts

Objective: In this study,by simulating the FSS physiological environment of osteoblasts in vivo,comparing the regulatory effects of unidirectional and oscillating FSS on osteoblast differentiation function,selecting an in vitro FSS cell culture model with significant promotion of osteoblast differentiation,further establishing a osteoblast culture model of PDK1 activator intervention under FSS,revealing the effect of the joint intervention on osteoblast differentiation and metabolism,and providing a basis for clinical motor rehabilitation and drug use of bone-related diseases.Methods: The experiment was mainly divided into two parts.The first part of MC3T3-E1 cell was routinely cultured,and the osteoblast cell line was identified by ALP staining and alizarin red staining;Screening of suitable hydrodynamic cell culture models by cytoskeleton fluorescence staining and relative quantitative PCR experiments.Unidirectional flow and oscillatory flow intervened in MC3T3-E1 cells were UF1 group and OF group,respectively.In the second part,after the intervention of PDK1 activator in MC3T3-E1 cells,relative quantitative PCR experiments,Western Blot experiments and flow cytometry were used to evaluate the role of unidirectional fluid shear stress and PDK1 in the differentiation of MC3T3-E1 cells.The experiment was divided into four groups,namely static group（group C）,static and PDK1 activator group（CP group）,Unidirectional flow group（UF2 group）and Unidirectional flow and PDK1 activator group（UP group）.Result:（1）The MC3T3-E1 cell line used in this experiment can form mature osteoblasts.（2）Compared with the UF1 group,the cells in the OF group showed a clustering phenomenon,and the F-actin fibers gathered into bundles,showing a stronger fluorescence intensity;the UF1 group had a higher expression of Runx2 m RNA than the OF group,and had Significant difference（P=0.0140<0.05）.（3）Compared with the C group,the CP group significantly increased the expression of Runx2 m RNA,and there was a significant difference（P=0.0346<0.05）;the CP group had more glucose intake than the C group,and there was a significant difference（P=0.0003<0.05）,there was no significant difference in mitochondrial mass（P=0.2810>0.05）.（4）The expression of Runx2 m RNA in the UP group was significantly higher than that in the UF2 group（P=0.0022<0.05）,the expression of MC3T3-E1 cells p-AKT in the UP group was more significant than in the UF2 group（P=0.0317<0.05）,and there was no significant difference in the expression of Runx2 and its downstream Osterix protein（P=0.1905>0.05;P=0.7302>0.05）;the UP group had more glucose intake and larger mitochondrial mass than the UF2 group,both of which had significant differences.（P=0.0022<0.05,P=0.0173<0.05）.（5）The results of PDK1 activator intervention index: the UF2 group had a greater effect on the expression of Runx2 m RNA in MC3T3-E1 cells than the C group（P=0.0152<0.05）;the UF2 group had less glucose intake than the C group,and there was a significant difference（P=0.0082<0.05）,the mitochondrial mass of UF2 group was significantly higher than that of C group（P=0.0221<0.05）.Conclusion:（1）Unidirectional flow and oscillatory flow affect the rearrangement of MC3T3-E1 cytoskeleton in different ways,and unidirectional flow can better promote osteoblast differentiation than oscillatory flow.（2）PDK1 activators can increase energy intake,thereby promoting osteoblast differentiation.（3）Unidirectional flow combined with PDK1 can simultaneously enhance the energy intake and mitochondrial level of MC3T3-E1 cells,and promote the differentiation of MC3T3-E1 cells to osteoblasts.（4）In the presence of PDK1,unidirectional flow promotes the differentiation of MC3T3-E1 cells by regulating the mitochondrial level.（5）Both unidirectional flow and PDK1 can promote MC3T3-E1 cells by regulating the energy metabolism of MC3T3-E1 cells Osteogenic differentiation of unidirectional flow,but unidirectional flow focuses on regulating aerobic metabolic capacity,whereas PDK1 is associated with energy intake,and PDK1 has a synergistic effect on the increase in mitochondrial levels after unidirectional flow interventions.