Abstract:

Objective To observe the influence of different doses of 17β-estradiol (17β-E2) on the osteogenesis differention of bone marrow mesenchymal stem cells (MSCs) and the expression of bone gamma-carboxyglutamic-acid-containing proteins (BGP) gene, and to clarify the mechanism of estrogen replacement treatment for postmenopausal osteoporosis. Methods The MSCs were isolated by density gradient centrifugation and adherence screening method for continuous three passages,and the osteogenic induction and differention were performed. The MSCs were divided into control group(OBM) and different doses of 17β-E2 groups(anding 0.1,1.0,10.0 and 100.0 pmol·L^-1 17β-E2 in osteogenic induction liquid).On the 7th day,radioimmunoassay was used to detect to alkaline phosphatase (ALP) activity,Von Kossa staining for calcium deposition of the cells in various groups.On the 14th day,the collagenⅠ (ColⅠ) level was determined by enzyme linked immunosorbent assay.On the 21th day,the BGP mRNA expression level was detected by real-time fluorescence quantitative PCR,and the cells'optical density (A) at 560 nm was determined. Results The MSCs were isolated successfully by density gradient centrifugation and adherence screening method.The MSCs appeared fibroblasts-like with ellipse nucleus with one to two nucleolus.The subculturing MSCs grew well and maintained the morphological characteristics of primary cells.Compared with control group,the ALP activities and Col Ⅰ levels in different doses of 17β-E2 groups were increased(P<0.05 or P<0.01),and the bone matrix calcification was enhanced in a dose-dependent manner (P<0.05 or P<0.01);the BGP mRNA expression level in 100.0 pmol·L^-1 17β-E2 group was increased(P<0.01). There were significant differences in the BGPmRNA expression levels difference between 10.0 and 100.0pmol·L^-1 groups (P<0.05) ALP. The col-I levels and bone matrix calcification had significant differences between various groups (P<0.05). Conclusion The separated cells in this study are the rat MSCs. 17β-E2 can enhance the osteogenesis differention of MSCs and up-regulate the BGP mRNA expression in a dose-dependent manner.

Key words: mesenchymal stem cells, oestrogen, osteocalcin, osteoblasts