Puerarin Induced Osteogenic Differentiation Of Adipose-derived Stem Cell In Postmenopausal Osteoporosis Rat

Objective: To explore the quantity and viability of Adipose-derived stem cell(ADSCs)in postmenopausal osteoporosis rats by in vitro experiments,and ADSCs osteogenetic differentiation effect in vitro induced by puerarin in postmenopausal osteoporosis rat.To explore the maximum effective concentration and time of puerarin induced osteogenic differentiation of ADSCs in postmenopausal osteoporosis rats.Methods: Experiment 1 using 30 female SD rats aged 3 months,randomly divided into three groups(10 in each): surgical remove bilateral ovaries(Ovariectomized,OVX)in postmenopausal osteoporosis rats model,at the same time set up the normal Control group(Control)and Sham group(Sham);The number and vitality of ADSCs in three groups were compared by the adherent culture method and Transwell method.Flow cytometry was used to identify the 3rd and 21 st passage ADSCs phenotypes and compare the results in OVX group.Experiment 2 Acquire the 3rd passage ADSCs in logarithmic phase of OVX group for nontoxic dose test,according to the results of the test respectively prepare puerarin concentration in high,medium and low group and negative control group,at the 7th day of cell culture use alkaline phosphatase staining and at 21 st day use alizarin red staining and analysis dyeing effect.The activity of alkaline phosphatase was detected at the 5th,7th,10 th and 12 th day of cell culture.Results: 1.after about 8-10 hours of primary cell culture of the three groups of ADSCs,most of the cells begin to stick to the bottom wall,and the cells can cover over 80% after 4-6 days,and can sub cultured after 7 days.After digestion,the growth rate of cells was significantly faster than that ofthe original passage,and cells could cover over 80% in 3-5 days.The MTT method was used to determine that there was no statistical difference in growth curves of the three groups(P>0.05).There was no statistically significant difference in cell adherent rate of the three groups(P>0.05).ADSCs migration ability test,no statistical difference was found between the three groups(P>0.05).ADSCs flow cytometry identify cellular immune phenotype,appraisal analysis of cell surface antigen,the CD29,CD44,CD90 express positive,CD45,CD11 B express negative,and the 21 st passage express the same antigen with those of the 3rd passage,not weaken along with the increase of passage,two sets of results with no statistical difference(P> 0.05).2.According to the nontoxic dosage assay below 5% of the cell damage rate,we prepare puerarin solution in 12?mol/ml,1.5?mol/ml,0.25?mol/ml,and negative control group in 0?mol/ml.After 7 days of culturing,the alkaline phosphatase staining show clear different degree of blue in three groups to high medium and low concentrations and no obvious blue in negative control group.After 21 days of cultivation,the red mineralized nodules were clearly observed in different degrees and the negative control group was not in red.Four groups of alkaline phosphatase activity assay found that puerarin can significantly affect the activity of alkaline phosphatase,the puerarin concentration and time were positively correlated with alkaline phosphatase activity(P<0.05)but the negative control group does not change over time in alkaline phosphatase activity(P>0.05).Conclusion: 1.this study isolated the ADSCs from postmenopausal osteoporosis rats and successfully cultured and proliferated in vitro to the 21 st generation or more,and there was no significant difference in the appearance of the subcells.Compared with the normal control group and the sham group,there was no statistically significant difference in the number and viability of ADSCs in osteoporosis group(P>0.05).The osteoporosis rats ADSCs can be used as the source of autologous stem cells for bone defect repair.2.Puerarin has the effect of inducing osteogenic differentiation of ADSCs in vitro,and its osteogenic differentiation efficiency is positively correlated with the concentration and time in the non-toxic dose.The maximum effective non-toxic dose of Puerarin is at the concentration of12?mol/ml.