microRNA在激素性股骨头坏死骨髓干细胞成骨分化中调控机制的研究

The exact pathogenesis of Glucocorticoid induced femoral head necrosis is still not clear.It has been confirmed that hormones can promote bone marrow mesenchymal stem cells adipogenic differentiation and inhibit the osteogenic differentiation, which induce or aggravate the bone avascular necrosis.Therefore, In recent years,therefore,the application of stem cells with osteogenic differentiation potential to treat avascular necrosis is becoming the hotspot, but its osteogenic regulation mechanism is not clear. The differentiation of stem cells regulated by microRNA has also been confirmed by a lot of studies.So we hypothesized that it is microRNA which be influenced by glucocorticoid regulates the differentiation of bone marrow mesenchymal stem cells, and disturbs the balance of the bone necrosis and repair.We will screen the differences of microRNA between steroid-induced avascular necrosis of the femoral head and normal human, through comparative analysis combined with bioinformatic prediction ,we hope to find key microRNA, and to verify its function by molecular biology techniques and animal models. Explore microRNA regulation mechanism of steroid-induced necrosis of femoral head, providing powerful evidencethe for clinical treatment.

糖皮质激素导致股骨头坏死的确切发病机制尚不明确，已有研究发现激素可以促进骨髓间充质干细胞成脂分化并抑制其成骨分化，从而诱发或加重骨坏死。因此近年来应用干细胞具有成骨定向分化的潜能，治疗骨坏死正成为该领域的热点，但其成骨调控机制并不清楚。而在干细胞的分化过程中，microRNA的调控作用已被很多研究证实。因此我们假设激素通过microRNA调控骨髓间充质干细胞的分化潜能而影响骨坏死的发生或修复。本研究拟筛选激素性股骨头坏死患者与正常人骨髓间充质干细胞表达的差异性microRNA，通过对比分析，联合运用生物信息学方法寻找调控股骨头坏死进程中影响干细胞分化的关键microRNA，采用分子生物学技术模拟其高表达或低表达对骨髓间质干细胞分化功能的影响并在动物水平进行验证，以探索激素性股骨头坏死的microRNA调控机制，为激素性股骨头坏死的发生机制和临床应用干细胞治疗提供有力的依据。

激素性股骨头坏死（ONFH）的发病机制尚不清楚，其发生发展可能与间充质干细胞（MSC）的成骨、成脂分化能力密切相关，而microRNA广泛参与MSC的成骨、成脂分化过程。在本研究过程中，我们首先分离正常MSC、股骨头坏死MSC和大剂量地塞米松刺激的MSC，提取RNA，通过基因芯片测定发现miR-423-5p、miR-193b、miR-744、miR-214、miR-423-3p、miR-320a、miR-140-3p等7个miRNAs表达下降，miR-152、miR-23a、miR-425、miR-27a、miR-221、miR-151-5p、miR-22、miR-224等8个miRNAs表达上调。通过RT-PCR验证miR-744、miR-149-3p、miR-23a和miR-320a表达情况与芯片结果保持一致。挑选miR-23a，将其mimics和inhibitor分别导入正常MSC，发现miR-23a抑制成骨分化而对细胞周期无明显影响；通过生物信息学分析对miR-23a的靶基因进行初步的预测，通过双荧光素酶实验对靶基因的结构匹配性进行验证，最终确定LRP5作为miR-23a靶基因；通过siRNA干扰技术干扰MSC中LRP5表达，发现其成骨能力增强，从而证明miR-23a通过影响MSC中LRP5表达抑制成骨分化过程。此后，我们制备大鼠激素性股骨头坏死模型及miR-23a mimics 和 inhibitor 慢病毒，并将慢病毒转染至大鼠MSC中，于造模4周后给大鼠注射转染的MSC，发现microRNA-23a在大鼠体内能够调控BMSC成骨分化能力，从而对大鼠激素性股骨头坏死呈现不同程度的修复效应。抑制microRNA-23a表达，股骨头坏死发生率降低，骨小梁参数BV/TV、Tb.Th升高，LRP5表达升高，micRNA-23a表达升高则呈现相反的效应。本研究证实了microRNA可以通过影响MSC成骨分化调控激素性股骨头坏死的发生发展，而miR-23a在体内、体外均通过LRP5抑制MSC成骨分化，抑制其表达可能对于坏死区域的修复具有促进作用，可能成为潜在的治疗靶点。