| PartⅠ Establishment and identification of osteoporotic mice model Objective: To establish the osteoporotic mice model.Methods: 30 10-week-old female C57BL/6 mice were randomly divided into 2 groups: ovariectomized group(OVX)and operation control group.Bilateral ovariectomy was performed in mice from OVX group while the others were not.At 6 weeks after the operation,femora from two groups were respectively collected to detect the bone mass by using micro CT analysis and HE and Masson staining.Results: Compared with the operation control group,a marked reduction in bone mass and trabecular bone volume,as well as the decrease of cortical thickness and the enlargement of the bone marrow cavity of mice can be seen in OVX group.Conclusion: The osteoporotic mice model was successfully constructed.PartⅡ Isolation,culturing and identification of ASCs from osteoporotic mice Objective: To isolate,culture ASCs from osteoporotic mice,and identify the acquired cells were OP-ASCs through checking the ability of multi-directional differentiation and the expression of surface antigens of the cells.Methods: Adipose tissues of inguinal region were obtained from osteoporotic mice in sterile condition,and the cells were obtained through the method of explanting culture.The ability of multi-directional differentiation of the cells were identified by alizarin red staining after 14 days of osteogenic induction and by oil red O staining after 21 days of adipogenic induction,and the expression of cell surface antigens such as CD14,CD34,CD44,CD90,CD105,Sca-1 were detected by flow cytometric analysis.Results: Acquired cells were adherent cells,fusiform or spindle shaped,sometimes arranged in clusters or swirls;mineralized nodules and oil droplets were successfully induced after separately culturing in osteogenic or adipogenic medium.The results of flow cytometry showed that the positive expression of CD14,CD34 was respectively 0.5%,0.59%,while then CD44,CD90,CD105,Sca-1 was respectively 97.01%,99.12%,95.19%,99.69%.Conclusion: The isolated cells were OP-ASCs.PartⅢ The effection of Wnt5 a overexpression mediated by lentivirus on osteogenic differentiation of OP-ASCs Objective: To clarify the effection of Wnt5 a on osteogenic differention of OP-ASCs.Methods: Construct a recombinant lentiviral vector overexpressing Wnt5 a,and OP-ASCs were infected with the lentiviral vectors in the condition of the best MOI,and screened by using puromycin,the mRNA and protein expression of Wnt5 a were detected by qPCR and Western blot.The total RNA and protein of three groups including overexpressing Wnt5 a group,the relevant empty vector group and the control group were respectively extracted after osteogenic induction 1 day,3 days and the expression of mRNA and protein of osteogenic related transcription factors such as OPN,Runx2 and ALP were respectively detected by qPCR and Western blot.Results: The MOI value of the Wnt5 a overexpression group was 80,the transfection rate was nearly 80%;the expression of Wnt5 a mRNA and protein in overexpression group was significantly higher than that in the relevant empty vector group and the control group(p<0.05);while the expression of osteogenic related transcription factors were lower than that in the relevant empty vector group and the control group after osteogenic induction 1 day and 3 days(p<0.05).Conclusion: Overexpression of Wnt5 a could inhibit the osteogenic differentiation of OP-ASCs. |
