Role Of Wnt Signaling In The Osteogenic Differentiation Of Adipose-derived Stromal Cells In Osteoporotic Mice

Part Ⅰ.Establishment of the osteoporosis mice modelObjective:To establish the osteoporosis（OP）mice model and provide the disease animal model for the follow-up study.Methods:A total of 60,4-week-old,C57BL/6 female mice were enrolled and randomized into test group（n=40）and control group（n=20）.Test group underwent bilateral OVX（OVX group）and control group was subjected to sham surgery（Sham group）.At 6weeks after surgery,3 animal in each group were randomly killed and the distal tibias were harvested.Micro-CT measurement was used for observing the average bone mineral density of the bone in the proximal tibia and the number of trabecular bone of the mice,and HE and Masson were used to observe the number and structure of the bone in the proximal tibia.Comparative analysis was used to evaluate whether the OP mice model was established successfully through results of Micro-CT and tissue sections of bone specimenin of Mice in OVX group and Sham group.Results:All the mice were survived after surgery and surgical infection or other complications did not happen.Micro-CT scan shown:the BMD、TV/BV and Tb.N in the proximal tibia of mice in OVX group were lower than that in the Sham group（P<0.05）;Tissue slice staining pointed:the number of trabecular bone in the proximal tibia in OVX group was reduced and the structure of trabecular bone was disorder compared with Sham group.Conclusion:OVX could make bone resorption obviously and the trabecular bone structure became loose and disorder.OVX was an effective method to establish the OP of animals and it could provide a stable and reliable model of OP mice for the follow-up study of this experiment.Part Ⅱ.Screening of Differentially Expressed Genes of Wnt Signaling Pathway in OP-ASCs and ASCsObjective:To compare the expression of Wnt signaling pathway in adipose tissue of OP mice and normal mice,and to explore the relationship between osteoporosis and Wnt signaling pathway.Compare the osteogenic potential of OP-ASCs and ASCs,and explore the significant difference genes related to Wnt,which can provide the foundation for the follow-up study on the relationship between Wnt signaling pathway and osteogenic ability of OP-ASCs.Methods:After the establishment of the OP mice model,3 mice were selected randomly by the envelope method from the OVX group and the Sham group.We Extracted of mRNA samples from adipose tissue on the right adrenal and the protein sample was derived from adipose tissue on theleft adrenal.The expression level of Wnt-related genes/proteins（β-Catenin and Gsk-3β）in adipose tissue of OVX group and Sham group were detected by qPCR and WB technique.Envelope method was employed to select mice randomly from OVX group and Sham group（n=30 and n=20,respectively）.The adipose tissue was took from bilateral groin and primary extraction as well as subculturing of ASCs and OP-ASCs were performed by tissue block method.P₃ OP-ASCs were used for osteogenic induction and it also used to explore the relationship between osteogenesis-related genes and induction time,which aimed at screen out the appropriate induction time.Osteogenic induction fluid was used to culture P₃ASCs and OP-ASCs and the osteogenic ability of ASCs and OP-ASCs was compared.The Wnt signal transduction gene was detected by P₃ ASCs and OP-ASCs,and the Wnt-related genes were screened out.The results were validated by qPCR and WB.Results:The results of qPCR suggested that osteogenesis-related genes（ALP and Runx2）were higher than before induction（P<0.05）on day 3 and day 5 and there was no significant difference in expression between day 3 and day 5,after the P₃ OP-ASCs cultured in osteogenic induction fluid 1,3,5days.The results of qPCR shown that:The expression of ALP,Runx2 and OPN in ASCs was higher than that in OP-ASCs（P<0.05）.The expression of Wnt5a,Fzd6 and Axin2 in ASCs and OP-ASCs was significantly different（P<0.05）,and the differences in expression between Wnt5a and Fzd6 were verified by qPCR and WB results,after the P₃ OP-ASCs and OP-ASCs cultured in osteogenic induction fluid 3days.Conclusion:Wnt signaling pathway in OP mice was lower than that in normal mice,which may be one of the causes of osteoporosis in OVX mice.The osteogenic differentiation potential of OP-ASCs is lower than normal ASCs,and Wnt5a and Fzd6 may play an important role.Part Ⅲ.Effect of LiCl and DKK-1 on the differentiation of OP-ASCs by Wnt signal pathwayObjective:To investigate the role of Wnt signaling pathway when the OP-ASCs differentiated into bone,and find a feasible method to improve the potential of OP-ASCs to differentiated into bone.Methods:On the basis of the second part,P3 OP-ASCs was took as the research object,LiCl and DKK-1 were used to adjust the signaling pathway expression level of Wnt in OP-ASCs,and the changes of Wnt correlation factor were detected by qPCR and WB technique.P₃OP-ASCs was selected to execute osteogenic induction when LiCl and DKK-1 were used to adjust the Wnt signal path,the expression of Wnt and osteogenesis-related genes and proteins was detected by qPCR,WB and immunofluorescence staining,alkaline phosphatase（ALP）staining and alizarin red（ALR）staining were used to observe osteogenesis.Results:After 3 days dealing with the LiCl,the qPCR and WB shown that:Wnt-related factorsβ-Catenin and Fzd6 increased（P<0.05）,but the expression of Gsk-3βand Wnt5a were decreased（P<0.05）.The LiCl and osteogenic induction were co-treated for 3 days,qPCR showed that:The expression of osteogenic genes（ALP,Runx2 and OPN）were higher than the control group（P<0.05）,WB showed:The expression of osteoblast-associated protein（Runx2 and OPN）were higher than that of the control group（P<0.05）,Immunofluorescence showed that the expression of Wnt-related proteinβ-Catenin and osteogenesis-associated protein Runx2 were increased.7 days and 21 days after dealing with the LiCl and osteogenic induction solution,both the ALP staining and ALR staining showed that the osteogenesis-related features were significantly higher than the control group.Conclusion:Inhibition of osteogenic differentiation potential of OP-ASCs could be reversed through activating the Wnt signal path by LiCl.Fzd6 and Wnt5a has undergone a corresponding change in the process of osteogenesis differentiation of OP-ASCs,which might be the key factor to recovery or promote the ability of OP-ASCs to differentiat into osteogenic,but its specific role and mechanism need to be explored in depth,and it can be the next research direction.