| Objective:To establish cultivation system in vitro of bone marrow stromal cells from SD rats,observe their growth and characteristics and investigate the effects of osthole on their proliferation and osteoblast-differentiation ability.And to investigate the expression of BMP-2mRNA in induced procedure with osthole.The study may provide a probable action mechanism of osthole prevent and cure OP in stem cell aspect.Methods:1.MSCs from SD rats were isolated and purified by using differential attachment method,then identified according to morphology,induced-differentiation potentiality and the cell surface antigens.So we can establish a stable culturing system in viro.2.MTT method was used to investigate the proliferation effects different concentrations Osthole on MSCs in SD rats,and blank control group.3.ALP-positive cell counts by the improvement Gomori's calcium-cobalt staining was explored to observe.Osteoblastic differentiation potentiality of MSCs induced by Different-concentration osthole.Blank control is standard medium without Osthole Through these we confirm the most effective Concentrations.4.Groups were divided:osthole group(induced by the most effective Concentrations),Blank control,classics group(classics induce bone formation group,positive control),Osthole +classics induce bone formation group;The ALP activity and the number of mineralized nodesls were measured to indicate the osteoblast function of different groups.5.Semi-quantitative analysis was used to detect the expression of BMP-2 during osteodifferentiation through RT-PCR method,Grouping was same as above.Results:1.Primary cultured MSCs adhered to plastic surface within 24 hours and reached confluence within 10-12days.To be conditioned induced,MSCs were successfully differentiated into osteoblast and adipocyte.And confirmed that there was expression of CD29 and CD44, and no expression of CD34 and CD45 on the surface of MSCs by flow cytometry(FCM).2.OD values of MTT shows Osthole groups of 1×10-5,1×10-6,1×10-7 mol/L were higher than blank groups,significantly(P＜0.05),especiallyl×10-6 mol/L. 3.Results of ALP staining for ALP-positive cell counts showed Osthole of 1×10-6mol/L addition had a definit induction to osteoblast differentiation compared with the blank with significant difference(P＜0.05).1×10-4,1×10-5,1×10-7,1×10-8 mol/L with nosignificant difference(P＞0.05).4.ALP activity values in MSCs 12 days of osthole groups were higher than that in blank groups. But there were no significant Differences between classics group and Osthole +classics induce bone formation group(P＞0.05).5.The average number of mineralized nodes in MSCs 21days of osthole groups were higher than that in blank group.But there were no significant differences between classics group and Osthole +classics induce bone formation group(P＞0.05).6.The expression of the BMP-2 mRNA was stronger than bank group.There were significant differences between Osthole group and classics group(P＜0.05).But there were no significant differences between classics group and Osthole +classics induce bone formation group P＞0.05).Conclusion:1.MSCs can be isolated from rat bone marrow by the method of Differential attachment.2.Osthole in all these concentrations of 1×10-5 mol/L,1×10-6 mol/L,1×10-7mol/L all can promote the proliferation of primary mouse bone MSCs3.Osthole in the concentration of 1×10-6 mol/L can induce differentiation to osteoblast of MSCs;can improve the activity of ALP and the number of mineralized nodes.4.Osthole in the concentration of 1×10-6 mol/L can enhance the expression of BMP-2 and mRNA,which was may be the mechanism of inducing differentiation to osteoblast of MSCs.
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