Primary Culture Of Mouse Metaphyseal Pericytes And Its Role In Osteoporosis

OBJECTIVE:To explore the method of inducing and purifying the microvascular pericytes in bone tissue,we cultured mouse long bone metaphyseal microvascular tissues in order to investigate how the metaphyseal pericytes participate in the pathological process of osteoporosis by affecting angiogenesis in bone tissue.Methods:A total of 20 healthy C57BL/6 mice aged 8 weeks were randomly divided into two groups.The OVX group underwent ovarian castration to establish an osteoporosis model,and the SHAM group was a pseudo-surgical group.Paraffin sections were prepared from metaphyseal bone tissue for immunofluorescence labeling(CD 146,PDGFR?,and NG2)and the distribution of microvascular pericytes on the metaphysis was observed.In the SHAM groups,the metaphysis of the femur and tibia was sliced,and the metaphyseal bone tissue containing microvessels were adherently cultured.After primary cultivation,use selective medium(pericytes medium)to induce pericytes and continue to subculture the cells.Use western blot to detect chondroitin sulfate glycoprotein 2(NG-2),platelet-derived growth factor receptor(PDGFR?)and CD 146 expression to identify whether it is consistent with pericyte characteristics.Flow cytometry and immunofluorescence staining technique were also used to identify cells after induction and purification.Results:Western blot and tissue immunofluorescence confirmed the presence of microvascular pericytes in the metaphysis of mice,and the number of microvascular pericytes in the metaphysis of the OVX group was significantly lower than that in SHAM group;Western blot test and tissue immunofluorescence also confirmed that the cells after induction positively express CD 146,PDGFR? and CD 146.Flow cytometry showed that CD31 was negatively expressed.The cells after induction and purification conformed to the characteristics of pericytes,and the cell properties were uniform and stable,with few endothelial cell contaminations.CONCLUSION:Metaphyseal pericytes can be cultured from the microvascular tissue of mouse long bong metaphysis by using commercial selective medium.At the same time,there was a significant difference between the number of metaphyseal pericytes in the OVX group and the SHAM group,providing the evidence for the metaphyseal pericytes participating in the pathological process of osteoporosis by affecting blood vessel formation in bone tissue.