Abstract: Objective:To observe the effect of 17β-estradiol (17β-E2) on the calcium (Ca²⁺) channels during the osteogenic differentiation of rat bone marrow mesenchymal stem cells (MSCs), and to elucidate the mechanism of 17β-E2 in the osteogenic differentiation of MSCs. Methods:The MSCs were separated by density gradient centrifugation and adherent screening, and passaged for 3 times continuously to induce osteoblast differentiation. The MSCs were divided into control group[cultivated in osteoblast culture medium alone (OBM)] and different doses of 17β-E2 groups(added with 0.1, 1.0, 10.0, and 100.0 pmol·L^-1 17β-E2 in OBM, respectively). On the 14th day of osteogenic induction, the cells in each group were stained with Fluo-3/AM, and the Ca²⁺ levels were determinated by laser scanning confocal microscope;the mean fluorescence intensity (MFI) was used to respresent the level of Ca²⁺. Whole-cell Ca²⁺ currents were recorded using whole-cell patch clamp technique under different conditions. Results:The MSCs with fibroblast-like cells, oval nuclei and visible nucleoli were successfully isolated by density gradient centrifugation and adherent screening. The subcultured MSCs grew vigorously and maintained the morphological characteristics of primary cells. Following the increase of 17β-E2 concentration,the Fluo-3 fluorescence staining intensity of Ca²⁺ in each group was also gradually increased, especially in 100.0 pmol·L^-1 17β-E2 group. Compared with control group, the MFI of Ca²⁺ and the current peak values of Ca²⁺ in 10.0 and 100.0 pmol·L^-1 17β-E2 groups were increased (P<0.05 or P<0.01) during osteogenic differentiation of the MSCs; the MFI of Ca²⁺ and the current peak values of Ca²⁺ in 0.1 and 1.0 pmol·L^-1 17β-E2 groups showed no significant differences (P>0.05). Conclusion:The cells isolated by density gradient and adherent screening method are the rat MScs. 17β-E2 plays a role in promoting osteogenesis by enhancing the opening of Ca²⁺ channels in the MSCs and the inward current of calcium ions in a dose-dependent manner.

Key words: mesenchymal stem cells, 17β-estradiol, calcium channels, osteoblasts