microRNA对糖尿病大鼠颌骨骨髓基质干细胞骨向分化调控作用的研究

Diabetes mellitus is a common metabolic disorder characterized by hyperglycemia.A complex syndrome with more than one cause, diabetes is responsible for numerous complications affecting the whole body.Diabetes can cause disorder of bone merabolism leading to bone loss or osteoporosis and jaw bones are also involved in. Moreover, compared to individuals without diabetes, patients with diabetes are more susceptible to periodontal disease. And subjects with moderate to severe alveolar bone loss have an increased prevalence in diabetes mellitus. Individuals with poorly controlled diabetes are more susceptible to develop complications after implant therapy compared to individuals with well-controlled diabetes.It has been reported that bone loss caused by diabetes was mainly due to decease in new bone formation.Bone mesenchymal stem Cells (BMSCs) provide an excellent source of proliferative osteoprogenitor cells that can be induced to differentiate into osteoblasts. However,the ability of proliferation, anti-apoptosis and osteogenic differentiation of BMSCs were greatly decreasd in high glucose microenviroments,but the exact mechanism that how diabetes induces bone loss has been poorly defined so far.MicroRNAs are single-stranded RNAs of about 22 nucleotides that are found in a wide variety of organisms, from plants to insects to humans and had an important role in self-renew and differentiation of multipotent mesenchymal stromal cells (MSCs).Therefore, we hypothesize that certain groups of microRNAs are regulated during osteogenic differentiation of BMSCs obtained from streptozotocin induced rats model of diabetes mellitus.We will screen and verify special microRNAs that are differential expression during osteogenic differentiation of BMSCs obtained from normal and diabetic rats mandibular.We will further find and verify the target gene and clarify the role of key microRNA in signaling pathway.Our aim is to explore the mechanism of diminished osteogenic capacity of BMSCs in high glucose microenvironment through microRNA and signaling pathway . This work will provide a new insight into bone metabolism of daibetes subjects and would be helpful to restore the alveolar bone in patients with diabetes in the future.

糖尿病(diabetes mellitus，DM)患者常伴有继发性骨量减少及骨质疏松等DM性骨病，患者颌骨也往往表现出相关变化，如牙槽骨的丧失及颌骨的骨质疏松。研究发现,作为成骨细胞重要来源的骨髓间充质干细胞（BMSCs）,在高糖环境下其增殖、抗凋亡以及成骨分化能力都会受到明显影响，但是具体分子生物学机制仍不清楚。microRNAs是一类广泛存在于各类生物体中的长约22个核苷酸的非编码单链RNA分子，对干细胞的自我更新和分化起着精细的调节作用，其作用的发挥易受到微环境的影响。因此，本实验从microRNAs和信号通路角度出发，研究糖尿病大鼠的颌骨BMSCs的生物学功能，探讨高糖微环境对大鼠颌骨BMSCs成骨分化的影响，筛选出并验证在此过程中差异表达的关键microRNAs，阐明其与信号通路的相互作用机制,为揭示糖尿病引起的牙槽骨丧失提供理论依据。

糖尿病(diabetes mellitus，DM)患者常伴有继发性骨量减少及骨质疏松等DM性骨病，患者颌骨也往往表现出相关变化，如牙槽骨的丧失及颌骨的骨质疏松。由于颌骨的质量是种植成功的保证，因此糖尿病患者缺牙种植修复的成功率一直低于正常人。如何提高糖尿患者的种植牙成功率一直是困扰临床医生的一个难题，阐明糖尿病患者的骨质丧失的机制无疑是解决该难题首要的一步。.本课题就糖尿病大鼠颌骨来源的骨髓间充质干细胞（BMSCs）的成骨分化进行探讨，发现与正常大鼠相比，糖尿病大鼠来源的BMSCs的成骨分化能力大大降低，即使在体外正常环境下培养，仍然不能彻底恢复其成骨能力，说明BMSCs具有某种记忆功能。通过对BMP信号通路中关键因子的检测，我们发现BMP信号通路的抑制在糖尿病大鼠来源BMSCs的成骨分化过程中扮演了重要角色。通过microRNA芯片筛选以及实时定量PCR验证，共发现有9个下调，10个上调的差异表达miRNA。在上调的miRNA中，我们发现miR-135a、miR-let-7b、miR-132-3p和miR-2895与成骨相关且差异倍数较大，通过进一步实验验证，我们发现miR-135a在成骨过程中持续表达，并且随时间逐渐降低，呈现出时间依赖性。通过靶基因预测和验证，发现BMP信号通路中的BMP的I型受体（BMPR1a）是其靶基因之一。在BMSCs成骨分化过程中过表达miR-135a，可以显著抑制BMPR1a 的活性，同时BMP信号通路中的关键因子Smad1、Smad5、Smad8以及Runx2的表达都出现下降，BMSCs的成骨分化受到抑制，表明miR-135a可以通过抑制BMPR1a而对BMP信号通路产生抑制，从而抑制BMSCs的成骨分化过程。对于糖尿病大鼠来源的BMSCs的成骨分化过程中转染miR-135a inhibitor可以使其成骨能力得到一定的恢复。本研究为糖尿病引起口腔牙槽骨丧失提供了一定的理论依据，为寻求糖尿病条件下口腔诊治的新策略提供理论和实验依据。希望对提高糖尿病患者种植牙成功率的药物研发上也有所帮助。