miR-210 介导的牙周膜干细胞修复标准骨缺损及ERK/P38信号转导通路的调控机制

The repair and reconstruction of critical size defects (CSD) is one of the major problems faced by the clinical medicine. Tissue engineering technology has been widely regarded as a hotspot for studying the repair of CSD. Previous studies showed that low oxygen can stimulate overexpression of miR-210 ; miR-210 is a multiple effect factor, and can not only promote the formation of blood vessels, but also enhance bone formation. The researchers found that hypoxia can enhance the osteogenesis of periodontal ligament stem cells (PDLSC) However, till now, the relationship of miR-210 and osteogenesis of PDLSC has remained unclaer. We hypothesize that miR-210 can induce PDLSC aniogenesis and osteogenesis. This hypothesis has been partly verified in the preliminary experiments. We found that miR-210 is highly expressed in miRNAs when PDLSC was cultured under hypoxia,and miR-210 can promote PDLSC double differentiation of angiogenesis osteogenesis for the first time. In view of above data, this work intends to establish the CSD animal models in rats and dogs, and to confirm our hypothesis on 4 different levels (i.e., mRNA, proteins, small animals, and large animals) via lentiviral vector transduction, gene chips detection, vascularized tissue bone construction, and dental implant. This study will elucidate the effect of using miR-210-mediated tissue engineered bone to reconstruct oral biological function of CSD and regulatory function of signal transduction pathway.

大量标准骨缺损(CSD)的修复与重建是临床难题之一。运用基因增强的组织工程技术修复CSD 成为当前研究热点。文献报道低氧可以刺激miR-210过表达；miR-210是多重作用因子，具有促进血管形成和骨生成的双重调控作用。研究者发现低氧促进牙周膜干细胞 (PDLSC) 骨向分化，然而是否与miR-210有关，迄今国内外尚未见报道。我们推测 miR-210具有促进 PDLSC血管及骨向分化作用。这种假说在预实验中得到初步验证，我们首次发现低氧培养PDLSC 时miR-210高表达且miR-210具有促进PDLSC血管及骨向分化作用。鉴于此，本项目拟构建大鼠及犬CSD动物模型，应用慢病毒载体转染、基因芯片检测、血管化组织工程骨构建及牙种植体植入等技术，从mRNA-蛋白-小动物-大动物4层次验证我们的假说，阐明miR-210介导的血管化组织工程骨对CSD生物功能重建作用及其信号转导通路调控作用。

大量标准骨缺损 (CSD) 的修复与重建是临床医学面临的难题之一。运用基因增强的组织工程技术修复 CSD 成为当前研究的热点。在本课题的资助下： (1) 我们进行了牙周膜干细胞的分离和培养，体外研究结果表明 miR-210 具有促进牙周膜干细胞骨向分化的作用，但体内异位成骨效果不佳。基于此，我们调整了课题方案，利用 BMSCs 代替牙周膜干细胞完成了本课题。(2) 体外实验结果表明 miR-210 具有促进 BMSCs 骨向分化作用，首次利用 miR-210 介导的 BMSCs 进行了异位成骨实验，并取得理想疗效。通过基因芯片技术及分子生物学手段，明确了miR-210 作用于靶基因 sclerostin 激活 BMSCs 骨向分化通路。(2) 课题组成功构建了大鼠颅骨及犬下颌骨标准骨缺损模型，这为标准骨缺损的研究提供了基础。(3) 体外构建 miR-210 介导的组织工程复合体，利用该复合体进行了大鼠颅骨及犬下颌骨标准化骨缺损的修复重建研究。研究结果表明：miR-210 介导的组织工程复合体具有修复标准骨缺损的作用，研究论文已投稿 (under review)。通过本课题的研究，我们明确了 miR-210 为促进 BMSCs 血管和骨向分化的双调控基因，并明确了其作用靶基因。一系列体内外研究结果表明 miR-210 构建的组织工程化骨具有修复标准骨缺损的作用，这为将来的临床转化应用奠定了坚实基础。