| Objective To observe the effect of different concentration of 17β-estradiol on osteogenic differentiation of rat bone marrow mesenchymal stem cells(BMSCs) and analyze the differences in the expression of osteogenic differentiation related signal transduction molecules in Wnt/β-catenin pathway. We explore the mechanism that 17β-estradiol regulats the rat BMSCs on osteogenic differentiation through canonical Wnt signaling pathway and provide theoretical basis which applies estrogen or selective estrogen receptor modulators(SERMs) to the treatment of postmenopausal osteoporosis(PMOP) in clinic.Method We isolated bone marrow mononuclear cells from 6-week-old male SD rats femur and tibia through whole bone marrow culture method and purified BMSCs through times of medium changing and controlling the time of trypsin digestion during subculture. We identified BMSCs through three characteristics:(1)Wall sticking characteristic.(2) Multiple differentiation potential: We respectively induced P2 cells osteogenic and adipogenic differentiation and set up blank control. We changed medium totally every 3 days, carried on alkaline phosphatase staining in 7 days after osteogenic induction, Alizarin Red S staining in 21 days after osteogenic induction, oil red O staining was performed in 14 days after adipogenic induction, and observed their differentiation potential.(3)Cell surface markers were detected by flow cytometry: CD34、CD44、CD45、CD105. The rat BMSCs that were successfully identified inoculate into 6-well plate and separately culture by grouping 0、10-5、10-6、10-7、10-8、10-9 mol/L when they were fused about 80%, the cells were treated with the corresponding gradient concentration of 17β- estradiol, and the DMSO treatment was used as the control group. Meanwhile, all groups were carryed on osteogenic induction and changed medium every 3 days. We processed alkaline phosphatase staining,quantitative analysis of alkaline phosphatase and extracted total protein in 7 days after osteogenic induction. The level of Wnt10b、β-catenin、LRP5 were measured by western-blot. Alizarin Red S staining was conducted in 21 days after osteogenic induction.Result The mononuclear cells in the bone marrow of rats were isolated by the whole bone marrow culture method, and the cells had great morphology and proliferation activity. Changing medium many times and controlling the time of trypsin digestion when passage can be purified BMSCs, and the purity of P2 cells could reach 95%. BMSCs could differentiate into osteoblasts and adipocytes, after induced by osteogenic and adipogenic induction respectively in P2 cells. The rat BMSCs was successfully identified by flow cytometry, and the cell uniformity was high. Gradient concentration(0、10-5、10-6、10-7、10-8、10-9 mol/L)17β- estradiol is used for the intervention when P2 cells were induced by osteogenic induction. 7 days after osteogenic induction, alkaline phosphatase staining and quantitative analysis showed that ALP expression amount increased gradually, and activity was enhanced with the increase of 17β- estradiol concentration, 10-5 mol/L group of which was the most active, and there was significant difference between each treatment group and blank control group. After 7 days of osteogenic induction, the expression of protein in each group was detected by Western-blot, result of which showed expression amount of Wnt10 b and β-catenin elevated with the increase of 17β-estradiol concentration, and 10-5 mol/L group was the highest, but there was no significant difference in the expression of LRP5 in each group. After 21 days of osteogenic induction, Alizarin Red S staining showed that the number of red brown mineralized nodules in each group also elevated with the increase of 17β-estradiol concentration.Conclusion Through the whole bone marrow culture method, the BMSCs are screened out with high purity, good homogeneity and strong ability of proliferation. BMSCs have multiple differentiation potential which can differentiate into osteoblasts and adipocytes under particular inducing conditions. BMSCs of rat can perform osteogenic differentiation that is induced by estrogen in a dose-dependent manner in vitro, and also involved in the differentiation and maturation of osteoblast. This effect may be achieved by up-regulating the expression of Wnt10 b and β-catenin in the canonical Wnt signaling pathway. However, estrogen has no significant effect on the expression of membrane receptor LRP5 in the canonical Wnt signaling pathway. |
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