| Objective: To investigate the role of canonical Wnt/?-catenin signaling pathway on osteogenic differentiation of adipose-derived stem cells from ovariectomized osteoporosis mice by establishing the animal model of osteoporosis mice,constructing overexpression and silenced lentiviral vectors specific to ?-catenin gene in vitro,up and down regulating the level of?-catenin expression of adipose-derived stem cells from ovariectomized osteoporosis mice.Methods: The 8-week-old female C57BL/6 mice(Weight:20g)were randomly divided into two groups: control group(sham opermion group)and experimental group(ovariectomized group).The experimental group was treated with bilateral ovariectomy,the control group removed the same amount of fat without ovariectomy.We collected right femurs from both groups of mice and detected the bone mass using micro-CT analysis at 6 weeks after operation.Besides,the changes of trabecular bone of right femurs were detected by HE staining and Masson staining.After successfully established the osteoporosis mice model,both the experimental group and control group of mice were sacrificed under anesthesia.Adipose-derived stem cells were obtained from inguinal adipose tissue of both groups.The isolated cells were cultured by tissue block method,and then purified and amplified to the third generation.The mesenchymal cell surface antigen of them was detected by flow cytometry(FCM).Immunofluorescence staining was used to detect Runt related transcription factor 2(RUNX2),Osteopontin(OPN),CCAAT/enhancer binding protein(C/EBP?)and Peroxisome proliferator-activated receptor gamma(PPAR?)at 7 days after osteogenic induction and adipogenic induction.Multilineage differentiation potential ofthese cells were detected using oil red O staining and alizarin red staining at 21 days after osteogenic induction and adipogenic induction.Constructing overexpression ?-catenin gene lentiviral vector and silenced ?-catenin gene lentiviral vector,and determinating the optimum MOI of adipose-derived stem cells from ovariectomized osteoporosis mice(OP-ASCs).OP-ASCs were infected with the most suitable MOI of lentivirus,and the infection efficiency was observed by immunofluorescence after 96 hours of infection.Screening of stably infected cells with puromycin,the relative mRNA expression and protein expression of ?-catenin/?-CATENIN was analyzed using quantitative RT-PCR and Western blot.The infected cells were induced to osteoblasts for 3and 5 days,and then the expression of osteoblastic markers(?-catenin/?-CATENIN,Runx2/RUNX2,Opn/OPN)were measured by Real-time PCR and Western blot.The infected cells were induced to osteoblasts for 10 days,alizarin red staining showed that mineralized nodules formation.Results: 1.Compared with the control group,the number of femoral trabeculae of the experimental group was significantly lower,normal structure disappeared,derangement distribution,expanded bone marrow cavity,these suggested that osteoporotic mouse animal models were successfully established.2.The results from FCM analysis showed these isolated cells were not contaminated by other cells such as monocytes or hematopoietic cells.Immunofluorescence staining was positive for RUNX2,OPN,C/EBP?and PPAR?.After 21 days of incubation in osteogenic and adipogenic medium,alizarin red staining and oil red O staining showed that mineralized nodules and lipid droplets formation.3.Overexpression ?-catenin gene lentiviral vector and silenced ?-catenin gene lentiviral vector were successfully established,determinated the two optimal MOI of infected OP-ASCs were 80.OP-ASCs can be stably infected by these lentiviruses.Real-time PCR and Western blot showed that the expression of ?-catenin/?-CATENIN,Runx2/RUNX2 andOpn/OPN was up-regulated in the overexpression group,the expression of these genes was down-regulated in the silenced group.4.Under osteogenic induction conditions,the expression of target genes and the formation of mineralized nodule in the overexpression group were significantly higher than those in the other two groups,the control group was the second,and the silenced group was the lowest.Conclusion: 1.The osteoporosis mice model was successfully constructed by ovariectomized method.2.OP-ASCs was successfully isolated and cultured by tissue block method.3.Overexpression?-catenin gene lentiviral vector and silenced ?-catenin gene lentiviral vector were successfully established.OP-ASCs can be stably infected by these lentiviruses.Wnt/?-catenin signaling pathway of OP-ASCs was successfully up-regulated and down-regulated.4.Wnt/?-catenin signaling pathway of overexpression group up-regulated enhanced OP-ASCs osteogenic differentiation ability.Wnt/?-catenin signaling pathway of silenced group down-regulated reduced OP-ASCs osteogenic differentiation ability. |
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