【作者】 王冰清；

【导师】 翁习生；

【作者基本信息】 北京协和医学院 ， 临床医学， 2013， 博士

【摘要】 研究背景目前,糖皮质激素被广泛应用于结缔组织疾病的治疗当中,大剂量糖皮质激素的应用常会诱发股骨头坏死,由激素使用引起的股骨头坏死在非创伤性股骨头坏死(osteonecrosis of femoral head, ONFH)的发病率中已经居于首位。股骨头坏死如果得不到有效治疗,将出现股骨头塌陷和骨关节炎,最终需行人工关节置换术,因此,早期发现股骨头坏死并行保头治疗显得非常必要。但由于股骨头坏死的病因还不是十分清楚,目前尚无有效治疗方法。基于病理研究,学者发现除微循环障碍导致的毛细血管的微栓塞引起局部骨内压增高,局部缺血坏死外,激素导致的骨髓间充干细胞成骨分化能力的改变,使得骨坏死和修复平衡打破,继而导致股骨头坏死塌陷也是发病机制重要的环节之一。microRNA是一种18—25nt长的单链小分子RNA,具有高度保守性、时序性和组织特异性,对于细胞组织的功能以及生命活动起着至关重要的调控作用,最新的研究发现microRNA在骨髓间充质干细胞的成骨分化中起着重要的调控作用。目前,激素刺激对于骨髓间充质干细胞的microRNA变化并不明确,在本实验中,我们通过建立小鼠股骨头坏死模型,分离培养其间充质干细胞,用microRNA芯片来寻找差异性表达的microRNA,通过生物信息学分析预测microRNA对基因的调控,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。研究目的1.建立激素性股骨头坏死的小鼠模型；2.寻找模型动物中BMSC的miRNA表达差异；3.分析表达差异的miRNA可能的调控作用；研究方法将6只小鼠随机分成2组,每组3只,实验组小鼠行甲基强的松龙21mg/kg皮下注射,连续4周,对照组同等剂量注射生理盐水。行股骨头切片,做HE染色及凋亡染色证明建模成功。取18只小鼠重新建模,做骨髓间充质干细胞原代培养,待其长至覆盖培养瓶80%后,流式鉴定,提取RNA,送检microRNA芯片,筛选差异性表达的microRNA,并进行生物信息学分析,获取此调控途径中的关键microRNA,预测其对于功能基因及信号通路的影响。研究结果1.较正常小鼠比较,激素刺激4周后,小鼠的股骨头切片出现了较多的空骨陷窝、骨小梁的断裂、纤维增生等改变。2.获取小鼠骨髓,做骨髓间充质干细胞培养,并通过形态及流式细胞技术鉴定,CD31-(96.04%)、CD34-(98.27%)、CD105+(95.13%)、CD166+(96.74%)3.正常组跟实验组在microRNA表达上存在差异,且在同种异体个体之间具有相对一致的表达趋势,其中microRNA-34b/c、microRNA-206、microRNA-148a、 microRNA-196a等16个microRNA在实验组表达下调明显,而microRNA-21microRNA-342、microRNA-92b等7个microRNA则在实验组高表达。4.在有共同表达趋势的microRNA中,选取感兴趣的microRNA进行生物信息学分析,发现microRNA-206、microRNA-34b、microRNA-34c、 microRNA-148a、microRNA-21、microRNA-652与成骨分化相关。结论1.激素性股骨头坏死可以引起骨髓间充质干细胞microRNA表达谱的改变；2.多种miRNA在这个过程中起到了调节作用,从本实验结果推测miRNA-196a下降在激素性股骨头坏死过程中起到了重要作用.更多还原

【Abstract】 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.更多还原