The Osteogenic Role Of Estrogen Receptor Alpha In Bone Marrow Mesenchymal Stem Cells

Understanding the mechanisms that control the proliferation and commitment of human stem cells into cells of the osteogenic lineage for the preservation of skeletal structure is of basic importance in bone physiology. Estrogen exerts its major long-term effects on cell growth, cell differentiation, and cell function via intracellular estrogen receptors. Eventually by activating particular genes, the cascade mechanism between ERαexpression and differentiation of bone marrow mesenchymal stem cells (BMSCs) is not clear, but the correlation between ERαexpression and BMSCs differentiation implies that E2 exerts its osteogenic effect through ERαas well as that dynamic loading induces bone formation requires ERαtoo.In this study, the primary cultured and identified female rat BMSCs were treated by various osteogenic methods, to observe the effects of estrogen and fluid shear stress on them. During the process many assay were performed, MTT, DNA contents, ALP activity and real time-PCR etc..The research consists the following objectives:1. to isolate and culture the BMSCs and identify them.2. to determine whether the Erαpresent in rat bone marrow mesenchymal stem cells (BMSCs).3. to evaluate the effects of 17β-estradiol (E2) on proliferation and differentiation in BMSCs.4. to identify whether fluid shear stress (FSS) is related with BMSCs'osteogenic commitment via ERα.5. to identify whether there is a synthetic effect of using the combination of FSS and E2.For in vitro experiments, the bone marrow was isolated from femurs and tibias of female SD rats. To test BMSCs'multipotential ability, Vacossa and oil red O staining were performed after specific inducing culture for osteogenesis and adipogenesis respectively. ERαwere assayed by western blotting, immunocytochemical staining, and further identified by real-time PCR. Erαwere proved presenting in the BMSCs. The effect of E2 on BMSCs proliferation was assayed by MTT test. Results showed that the proliferative activity was significantly promoted in BMSCs. The effect of E2 on differentiation of BMSCs was studied by using alkaline phosphatase (ALP) as a marker for the phenotype of mature osteblast.These cells were able to form both calcified nodules and adipocytes when they were cultured in charcoal-stripped fetal bovine serum (FBS). In our study, 10-6M E2 stimulated BMSCs proliferation and ERαexpression and the differentiation of progenitor cells into osteoblasts. 12 dyne/cm2 FSS had the similar effects on BMSCs osteogenisis. As the antagonist of ER, ICI182780 counteracted the above effects. Furthermore, the combination of the above mentioned E2 and FSS did not show the expected synthetic results.In conclusion, ERαexists in primary cultured rat BMSCs and its mRNA temporarily expressed with the culture stage of BMSCs. E2 enhanced ALP activity and increased ERαexpression. the effects of estrogen on osteogenesis in rat BMSCs is an ER-dependent way. FSS was also able to enhance the osteogenic commitment of BMSCs. No synthetic effect of the enhanced osteogenic commitment of BMSCs from the combination of FSS and E2.