人激素性股骨头坏死骨髓间充质干细胞差异表达miRNA筛选与miRNA-23a抑制干细胞成骨分化及机制研究

发布时间：2018-05-07 01:21

本文选题：激素性 + 股骨头坏死　；参考：《北京协和医学院》2015年博士论文

【摘要】：研究背景及目的激素性股骨头坏死(steroid-induced femoral head necrosis, SFHN)是由于长期使用激素类药物,导致以股骨头血供受损,骨细胞及骨髓成份死亡,继而导致股骨头结构改变、股骨头塌陷、关节功能障碍为特征的代谢性疾病。大约75%的患者从诊断SFHN起3年内便可发生股骨头塌陷,最终超过65%的患者不得不接受髋关节置换手术,给患者家庭和社会带来了巨大经济负担。探索SFHN的发病机制,从而及时有效地干预SFHN的进展将具有极大的经济效益和社会效益。SFHN发病机制的研究,国内外均未取得突破性进展。近年来,国内外学者对SFHN的发病机制进行了深入的研究,并提出了众多学说,其中包括骨内高压学说、凝血机制改变学说、脂肪栓塞学说、骨质疏松学说、骨细胞凋亡学说、膜微粒学说等等。至今,SFHN具体的发病机制仍不清楚,尚需进一步的研究。骨髓间充质干细胞(bone marrow mesenchymal stem cells, BMSC)是骨髓中一类具有多向分化潜能并在多种疾病发生发展过程中具有重要病理生理作用的细胞。体外实验发现大剂量糖皮质激素可通过糖皮质激素受体(GR)和激活蛋白-1(AP-1)途径抑制BMSC的增殖和成骨分化,相关研究也发现股骨头坏死患者转子部位成骨细胞的增殖能力下降。nicroRNA (miRNA)作为细胞内一类重要的非编码调控分子,它所介导的调控作用已经被认为是多细胞生物基因表达转录后水平的一种普遍调节方式。许多niRNA已被证明参与了骨质形成的过程,并在其中发挥关键作用。BMSC结合miRNA是研究SFHN发病机制的一个新的视角,存在为SFHN提供早期诊断与治疗的潜在价值。本课题将应用niRNA芯片筛选出调控SFHN相关的miRNA;对于我们筛选出的目标miRNA,进一步验证其功能,然后应用双荧光素酶报告基因系统、siRNA等技术探讨其调控机制,以期阐明激素性股骨头坏死的发病机制。方法1、BMSC的分离、培养与鉴定：从3例健康者和5例激素性股骨头坏死并接受外科手术治疗的患者体内取出骨髓,按照已建立的密度梯度离心法,获取BMSC,并培养传代,并对细胞表型、成骨等功能活性进行了初步的检验。2、差异性miRNA的筛选：在成骨分化过程中,对正常BMSC、激素性股骨头坏死BMSC和大剂量地塞米松刺激的BMSC提取RNA,然后通过基因芯片的方法测定miRNA表达情况,并筛选出差异性miRNA.3、差异性表达miR-23a的功能验证：将miR-23a的mimics(?)inhibitor导入正常BMSC,模拟过表达miR-23a和低表达miR-23a,然后进行成骨诱导,在3天、6天、9天和12天进行碱性磷酸酶染色、茜素红染色、Real Time PCR、western blot及ALP活性检测,进一步验证候选miR-23a对成骨作用的影响。4.miR-23a抑制BMSC成骨分化的机制：首先通过专业的靶基因预测网站,对候选miR-23a的靶基因进行初步的预测,然后通过双荧光素酶实验,验证靶基因与miR-23a的结构匹配性,最后通过siRNA干扰技术,进一步验证靶基因。最后通过通路抑制剂干扰具体的信号通路,验证miR-23a信号传导的具体通路。结果1、成功分离出BMSC,并通过流式细胞仪对其表型进行了鉴定,并通过相关染色、Real Time PCR的方式鉴定了其潜在的成骨分化能力。2、大剂量地塞米松刺激BMSC和激素性股骨头坏死B MSC中,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.3、选取miR-23a进行功能试验。细胞水平、蛋白水平和mRNA水平显示,miR-23a抑制成骨,结果与上述芯片结果相一致。4、通过TargetScan.miRBase等靶基因专业预测网站,预测8个候选靶基因；然后通过双荧光素酶报告实验,最终确定miR-23a靶基因为LRP5。最终确定miR23a通过Wnt通路抑制BMSC成骨分化。结论1、筛选出SFHN特异性miRNA表达谱。2.miR-23a靶基因为LRP5,通过Wnt通路抑制人BMSC成骨分化。
[Abstract]:Background and objective steroid-induced femoral head necrosis (SFHN) is a metabolic disease characterized by the damage of the blood supply of the femoral head, the death of bone cells and bone marrow components, and a metabolic disease characterized by structural changes of the femoral head, collapse of the femoral head, and joint dysfunction. About 75% of these are due to the long-term use of hormone drugs. The patients can collapse of the femoral head within 3 years from the diagnosis of SFHN, and in the end more than 65% of the patients have to accept hip replacement, which has brought a huge economic burden to the family and society. To explore the pathogenesis of SFHN and thus effectively intervene in the progress of SFHN in time will have great economic and social benefit.SFHN pathogenesis. In recent years, domestic and foreign scholars have not made a breakthrough at home and abroad. In recent years, domestic and foreign scholars have conducted in-depth studies on the pathogenesis of SFHN, and put forward numerous theories, including the theory of intraosseous hypertension, the theory of change of coagulation mechanism, the theory of fat embolism, the theory of osteoporosis, the theory of bone cell apoptosis, the theory of membrane particles and so on. So far, the specific SFHN is specific. The pathogenesis is still unclear, and further research is needed. Bone marrow mesenchymal stem cells (BMSC) is a kind of cells with multiple differentiation potential in the bone marrow and has important pathophysiological functions in the process of various diseases. In vitro, large doses of glucocorticoids can be used through sugar skin. The hormone receptor (GR) and activation protein -1 (AP-1) pathway inhibits the proliferation and osteogenic differentiation of BMSC. Related studies have also found that the proliferation of osteoblasts in the rotors of the femoral head necrosis patients decreased.NicroRNA (miRNA) as one of the most important non coding regulatory molecules in the cell. Its regulatory role has been considered to be multicellular organisms. Many niRNA have been proved to be involved in the process of bone formation, and the key role of.BMSC binding miRNA is a new perspective in the study of the pathogenesis of SFHN, and there is a potential value for the early diagnosis and treatment of SFHN. This topic will use niRNA chip to screen out the tone. Control SFHN related miRNA; for our screened target miRNA to further verify its function, and then apply the dual luciferase reporter gene system, siRNA and other techniques to explore the mechanism of its regulation in order to elucidate the pathogenesis of steroid necrosis of the femoral head. Methods 1, BMSC isolation, culture and identification: from 3 healthy persons and 5 cases of hormone femoral head. The bone marrow was removed from the patients with necrotic and surgical treatment. BMSC was obtained according to the established density gradient centrifugation, and the generation was cultured. The functional activity of cell phenotype and osteogenesis was preliminarily tested.2, differential miRNA screening: in the process of osteogenesis, normal BMSC, Hormonal Avascular Necrosis of the femoral head BMSC and large dose RNA was extracted from BMSC stimulated by dexamethasone, then the expression of miRNA was measured by gene chip method, and the functional verification of differential expression of miRNA.3 was screened and the differential expression of miR-23a was verified: miR-23a mimics (?) inhibitor was introduced into normal BMSC to simulate overexpression miR-23a and low expression miR-23a, and then osteogenic induction was performed at 3 days, 6 days, 9 days, and 12 days of alkaline phosphatase staining, alizarin red staining, Real Time PCR, Western blot and ALP activity detection, further verify the effect of candidate miR-23a on the osteogenesis effect of.4.miR-23a inhibition of BMSC osteogenesis differentiation mechanism: first of all through professional target gene prediction site, the candidate miR-23a target gene is preliminarily predicted, and then through double The luciferase test was used to verify the structure matching of the target gene and miR-23a. Finally, the target gene was verified by siRNA interference technique. Finally, the specific pathway of miR-23a signal transduction was verified by the pathway inhibitor interference specific signal pathway. Results 1, the BMSC was successfully isolated and the phenotype was identified by flow cytometry. The potential osteogenic differentiation ability of.2 was identified by Real Time PCR, and large dose of dexamethasone stimulated BMSC and hormonal necrosis of the femoral head necrosis, B MSC, miR-423-5p, miR-193b*, miR-744, miR-214, miR-423-3p, etc. -22, miR-224* and other 8 miRNAs expressions were up regulated and selected to perform functional tests. Cell level, protein level and mRNA level showed that miR-23a inhibited osteogenesis, and the results were consistent with the results of the above chip, and 8 candidate target genes were predicted through the professional prediction of the target gene of TargetScan.miRBase and other target genes. Then the double Luciferase Report was used to report the experiment. Finally, the miR-23a target base was determined because LRP5. ultimately determined that miR23a could inhibit BMSC osteogenesis through the Wnt pathway. Conclusion 1, the SFHN specific miRNA expression spectrum.2.miR-23a target group was screened by LRP5, and the BMSC osteogenic differentiation was inhibited by Wnt pathway.

【学位授予单位】：北京协和医学院
【学位级别】：博士
【学位授予年份】：2015
【分类号】：R681.8

【参考文献】