The Mechanism Of Microrna Regulation On Human Bone Marrow Stromal Cell Osteogenesis Treated By High Concentrations Of Dexamethasone

BackgroundNowdays, The ratio of steroid-induced osteonecrosis of the femoral head (ONFH) had elevated to the first rand of nontraumatic osteonecrosis of femoral head, and the patients were younger than before, the disability rate was high, which caused a great economic burden to the society. However, the etiology is still not very clear. Except the microthrombus caused by micro-circulation disorder which lead local intraosseous hypertension and ischemic, differentiation ability of bone marrow-derived mesenchymal stem cells changed by the glucocorticoids is another reason, which disturbs the balance of the bone necrosis and repair, resulting in femoral head collapse.With the development of molecular biology and epigenetics, the differentiation of stem cells regulated by microRNA has also been confirmed by a lot of studies. So we hypothesized that it is microRNA influenced by glucocorticoid regulates the di fferentiation of bone marrow mesenchymal stem cells. We will screen the differences of microRNA in BMSCs treated by different concentration of dexamethasone, through comparative analysis combined with bioinformatic prediction, we hope to find key microRNA, and to verify its function by molecular biology techniques. Explore microRNA regulation mechanism of steroid-induced necrosis of femoral head, providing powerful evidencethe for clinical treatment.Objects1. To isolate, culture and identy the bone marrow mesenchymal stem cells;2. To establish the cell model treated by high concentration of dexamethasone;3. To identify the different effect on bone marrow mesenchymal stem cells between different concentration dexamethasone;4. To screen the microRNA differences of bone marrow mesenchymal stem cells treated by different concentrations of dexamethasone; through comparative analysis combined with bioinformatic prediction, to find key microRNA;5. To verify microRNA function by molecular biology techniques.MethodsBone marrow was isolated from the proximal femur of three individual donors, who underwent total hip replacement. Human MSCs were isolated and cultured according to procedures approved by the Ethics Committee at the Chinese Academy of Medical Sciences and Peking Union Medical College. BMSCs were plated at a cell density of2×105cells in25-cm2, at80%confluence, medium was replaced with high osteoblasts-specific induction medium to induce the differentiation. ALP staining and Alizarin Red staining were maded at different time. MSCs were treated by different concentration of dexamethasone for48hours, without osteoblasts-specific induction medium. Total RNA was isolated from cultured cells. Screen different microRNA by gene chips, obtain the key microRNA through bioinformatics analysis, and verify its functional.Results1. Bone marrow mesenchymal stem cells have been successfully isolated, and have been confirmed by the cell morphology, capacity of osteogenic and adipogenic differentiation, and cell phenotypic identification.2. Compared with10-9mol/L Dex group, the proliferation and osteogenic differentiation of hBMSC are declined in10-'mol/L group, but adipogenic differentiation is much more obvious with the extension of time. Osteogenic gene test showed that:cultured with different concentrations of dexamethasone for12days, expression of osteocalcin, osteopontin, Runx2in10-9mol/L group is higher than that of10-7mol/L, the difference is significant (P<0.05).3. The different profile of miRNA is caused by different concentrations of dexamethasone, and it has relatively consistent expression trend in allograft. It shows:miRNA378d/f/g, miRNA1268a/b and miRNA196a/b in10-7mol/L are increased, but miRNA422a, miRNA378h/i/c and miRNA29a in10-9mol/L group are increased. 4. The putative target genes are predicted by Bioinformatics analysis, miRNA378, miRNA23, miRNA29are predicted to target BMP2, Runx2, DUSP2, which are related with osteogenic differentiation.Conclusion1. Human bone marrow mesenchymal stem cells can be separated and purified in vitro, hBMSC possess their unique cytologic morphology, cell surface markers and self-renewal and multilineage differentiation capacity.2. Compared with10-9mol/L dexamethasone, bone marrow mesenchymal stem cells proliferation and osteogenic differentiation is obvious inhibited by10-7mol/L.3. miRNA expression profile of bone marrow mesenchymal stem cell can be changed by different concentrations of dexamethasone; and the miRNA changed has correlation with osteogenic differentiation.