The Change Of MicroRNA Expression On Bone Marrow Stromal Cell In Mice With Steroid-induced Osteonecrosis Of Femoral Head

BackgroundNowdays, the glucocorticoid is widely used in the treatment of connective tissue disease, and the large dose of glucocorticoid may induce osteonecrosis of the femoral head. The ratio of steroid-induced osteonecrosis of femoral head (ONFH) had elevated to the first rand of nontraumatic osteonecrosis of femoral head. If the patient with ONFH can not reach the effective treatment, the femoral head will collapse and the osteoarthritis will come out, and the patients will need the operation of artificial joint replacement. So, the early diagnosis and treatment of ONFH is necessary. However, the etiology is still not very clear, the effective treatment of ONFH is not comfirmed. 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.MicroRNA is a small single stranded RNA whose length is about18-25nt,with highly conserved, time sequence and tissue specificity, and it plays a vital role in regulating the function of cell and tissue as well as in the regulationg of life activities. The latest studies found that microRNA plays a vital role in regulating the osteogenic differentiation of bone marrow mesenchymal stem cells. Currently, the microRNA change of bone marrow mesenchymal stem cells induced by glucocorticoid is not very clear. In this research, we established a mouse model of femoral head necrosis, isolated and cultured of mesenchymal stem cells, screened the differences of microRNA in BMSCs, and predicted its gene regulating through bioinformatic analysis. Explore microRNA regulation mechanism of steroid-induced necrosis of femoral head, providing powerful evidence for clinical treatment.Objects1. To establish a mouse model of femoral head necrosis; 2. To screen the microRNA differences of bone marrow mesenchymal stem cells in normal group and glucocorticoid-stimulated;3. To explore the relationship between microRNA and the osteogenic differentiation of BMSCs through bioinformatic analysis;Methods6mice are divided into2groups at random (normal group and experimental group), every group has3mice. The mice in the experimental group are subcutaneous injected for4weeks with21mg/kg methylprednisolone, and the mice in the normal group are injected with the same dose of normal saline. Then slice the femoral head, do HE staining and tunnel staining to make sure the model successe. Then rebuilt the animal model, isolate the bone marrow, and MSCs were isolated and cultured with whole marrow method. When BMSCs are at80%confluence, the BMSCs are comfirmed by flow cytometry. Then 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. Compared with normal group, after stimulated with MPS for4weeks, we can find much change in sections of femoral head, such as empty lacunas, fracture of bone trabecula, fibroplasia, and so on.2. Bone marrow mesenchymal stem cells have been successfully isolated, and have been confirmed by the cell morphology and flow cytometry, CD31-(96.04%)、CD34-(98.27%)、CD105+(95.13%)、CD166+(96.74%)3. The different profile of microRNA is caused by MPS injection, and it has relatively consistent expression trend in allograft. It shows:in experimental group, microRNA34b/c, microRNA206, microRNA148and microRNA196a are decreased, but microRNA21, microRNA342and microRNA92b in experimental group are increased.4. The putative target genes are predicted by Bioinformatics analysis, microRNA206、microRNA34b、microRNA34c、microRNA196a、microRNA148a、 microRNA21、microRNA652are related with osteogenic differentiation.Conclusion1. The steroid induced osteonecrosis of femoral head may cause microRNA changes in BMSCs。2. many miRNA regulate the process of osteogenic differentiation, and miRNA-196a decreasing take an important part in steroid induced osteonecrosis of femoral head.