Runx2/Dicer/miRNAs靶向调控糖尿病骨种植体界面成骨分化与骨整合的作用与机制研究

Dental implant is an important way to repair missing tooth, but the abnormal bone quality caused by diabetes can greatly ruin the implant health. Therefore, how to improve the success rate of dental implant in diabetes patients has become a key and difficult research topic. This project was designed to study the mechanisms in vitro and explore a possible targeting therapy in vivo. Biological function change of BMSCs cultured in different glucose concentration will be observed and analyzed. Specifically expressed gene and miRNAs in the diabetic model will be detected by using RT- PCR, Western blot and Northern blot.The process change of osteogenesis in BMSCs transfected with Runx2 and cultured in high glucose concentration will be evaluated. Bone metabolism genes and Dicer, which is the central enzyme to miRNAs, will be determined by RT- PCR, Western blot and luciferase analysis during osteogenesis.Gain-and-loss experiment is designed to disclose the function change of BMSCs during osteogenesis when enhancing or silencing Runx2.CHIP together with transfection technology will be applied to disclose whether Runx2 can bind Dicer promoter, thus trigger the lineage-Specific regulation of Runx2/Dicer/miRNAs during the osteogenesis and osseointegration process at the interface of bone-implant in diabetes mellitus disease. Furthermore, a kind of double membrane system containing PRF and BMSCs transfected with Runx2 will be constructed then implanted at the interface of bone-implant in diabetes rats, and the effect of this targeting therapy will be assessed. The expected results will help to reveal the mechanism that influent the osteogenesis and osseointegration process at the interface of bone-implant in diabetes mellitus，optimize the implant program in clinical diabetes patients by the application of tissue engineering technology, and has a broad clinical practice prospect and research value.

种植牙是修复缺失牙的重要方式，但糖尿病所致骨骼异常变化却极大危害了种植牙的健康，如何提高糖尿病患者种植牙成功率已成为当前研究的重点和难点。项目采用体外实验探讨机制结合体内实验模拟治疗并评估疗效的技术路线。首先检测高糖环境下BMSCs生物学功能变化，通过基因转染Runx2观察其在体外促进高糖环境下BMSCs成骨分化的能力;随后应用荧光素酶检测、Gain-and-loss实验、CHIP验证Runx2与Dicer启动子结合并影响下游成骨关键miRNAs，探讨Runx2/Dicer/miRNA促进成骨分化的机制；最后建立"糖尿病大鼠骨种植体"实验动物模型，构建转染Runx2的BMSCs/PRF双膜复合体植入糖尿病大鼠种植体骨界面，检测其促进成骨分化与骨整合的作用。项目结果有助于揭示糖尿病影响种植体骨界面骨向分化和骨整合的机制，并应用组织工程技术优化糖尿病患者种植方案，具有临床应用前景和研究意义。

糖尿病所致的成骨异常使口腔缺失牙的种植治疗面临挑战。课题组建立“糖尿病大鼠骨种植体”实验动物模型，发现在糖尿病大鼠股骨种植体周围，新骨形成速度和质量明显低于正常对照组。体外模拟高糖状态下成骨诱导培养BMSCs，发现随着糖浓度升高,细胞增殖能力增强，矿化能力降低，成骨能力降低。PI3K/AKT信号通路是胰岛素信号通路的核心部分，课题组发现PI3K/AKT通路与经典成骨信号Wnt/β-catenin通路可以经GSK3β产生相互效应，高糖环境可通过PI3K/AKT通路使p-GSK3β降低，抑制Wnt通路中β-catenin表达，使BMSCs成骨能力下降。当构建Runx2过表达质粒转染进BMSCs后发现，AKT、GSK3β、β-catenin磷酸化蛋白表达量和比例上调；而应用PI3K/AKT通路抑制剂LY294002可使这种效应减弱。亚细胞定位显示高糖环境下β-catenin大部分集聚在细胞质中，转染外源性Runx2质粒可促进β-catenin累积入核。Dicer在成骨分化过程中表达增高，且与Runx2表达趋势一致。课题组将包含Runx2结合位点的小鼠Dicer基因启动子片段插入到pGL3-basic报告基因质粒中构建pGL-Dicer promoter。双荧光素酶报告基因检测结果发现在HEK293细胞中Runx2可有效激活Dicer启动子的转录活性，且这一作用具有剂量依赖性；在MC3T3-E1细胞中siRNA干扰Runx2可抑制Dicer启动子的转录活性，证实了Dicer启动子区域包含Runx2转录所需的结合位点并具有转录激活功能。随后课题组通过染色质免疫共沉淀实验证实Runx2通过靶向结合miRNA关键酶Dicer启动子调控Dicer在成骨分化过程中的表达。在干扰Dicer基因表达的MC3T3-E1细胞中同时转染miR21 mimics或加入PTEN抑制剂VO-OHpic，均可部分逆转由于Dicer干扰引起的成骨分化抑制，提示miR-21a-5p可能通过靶向抑制PTEN参与调节成骨分化。课题组建立“beagle犬颌骨种植体周极限骨缺损”实验动物模型，应用脂肪干细胞细胞膜片复合PRF膜体外制备双膜复合体植入种植体周围，发现能有效促进新骨形成，改善骨结合。项目结果在一定程度上揭示了糖尿病影响干细胞成骨分化以及种植体周骨界面骨整合的机制，为转化应用组织工程技术提供新思路。