Effect of 17β-E_2 on Proliferation,Apoptosis and Cell Cycle of Osteoblasts Induced by BMSCs in Chicken Embryos

To investigate the effect of 17β-E2on osteoblasts induced by chicken bone marrow mesenchymal stem cells(BMSCs),the method of invitroculture of BMSCs and its inducement of osteoblasts were established in this experiment.Chicken BMSCs were purified with whole marrow adherent culture and subculture in vitro.The third generation of cells of BMSCs were selected for inducing osteoblastic culture.The growth and morphology,the cell proliferation rate and the cell cycle of osteoblasts induced-BMSCs were detected with inverted phase contrast microscope,methyl-thiazol-tetrazolium(MTT)and flow cytometry,respectively.Induced-BMSCs obsteoblasts were identified by alkaline phosphates(ALP),Van-Gieson staining and Alizarin red staining,respectively.The induced osteoblasts were divided into 5experimental groups and cultured with osteogenic differentiated medium containing different 17β-E2 concentrations of 10-7,10-8,10-9, 10-10,10-11 mol·L-1 for each group,respectively.Cells cultured without 17β-E2as a blank controlgroup.Effects of different concentrations of 17β-E2on the proliferation and differentiation of induced-BMSCs osteoblasts were measured by MTT and PNPP for finding the most effective concentration.Then this effective concentration of 17β-E2 was used for culturing induced-BMSCs osteoblasts,and the percentages of apoptotic cells were measured by flow cytometry.The results showed that BMSCs could be purified with whole marrow adherent culture.Osteoblasts could be induced from purified BMSCs using classic differentiated medium and were typically characteristic of the osteoblastic biology.The induced-BMSCs osteoblasts' ALP,collagen typeⅠand calcified nodules' staining were strongly positive compared with control group.10-9 mol·L-117β-E2concentration represented the most action of promoting proliferation and differentiation and inhibiting apoptosis of induced-BMSCs osteoblasts among the other concentration groups.Thus,it is shown that osteoblasts can be induced from purified chicken BMSCs and estrogen can enhance the function of induced-BMSCs osteobalsts.