miR-21介导的血管化组织工程骨对放射性骨坏死生物功能重建作用及HIF-1α/VEGF通路的调控机制

Jaw osteoradionecrosis (ORN) is a common complication of radio-therapy during head and neck tumors treatment. Meanwhile, low oxygen levels, low blood supply, and low cell counts are often observed in ORN area, resulted with significant challenges to the functional reconstruction of patients who suffer from severe deformities or jaw defects caused by ORN. Previous studies showed that miRNAs play an important role during the procedure of angiogenesis and vascularization. The researchers found that miR-21 is closely related with growth of blood vessel and this process mainly depends on HIF-1α/VEGF pathway to complete revascularization. However, till now, the relationship of miR-21 and bone formation has not been reported clearly. We hypothesize that miR-21 is a multiple effect factor, and can not only promote the formation of blood vessels, but also enhance bone formation. This hypothesis has been partly verified in the preliminary experiments. We found miR-21 can promote bone marrow mesenchymal stem cells double differentiation of angiogenesis osteogenesis for the first time. In view of above data, this work intends to establish the ORN animal models in rats and goats, 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-21-mediated tissue engineered bone to reconstruct oral biological function of ORN bone defects and regulatory function of signal transduction pathway.

颌骨放射性骨坏死(ORN)是头颈肿瘤放疗后常见的一种并发症，放疗区的组织内常出现"低氧、低血运、低细胞"现象，这使 ORN 性颌骨缺损的修复重建面临严峻挑战。文献报道 miRNAs 在血管发生及形成中起重要作用。研究者发现 miR-21与血管生长密切相关，其通过HIF-1α/VEGF通路完成血管再生。但是，miR-21与骨形成的关系，迄今国内外尚未报道。我们推测 miR-21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。这种假说在预实验中得到初步验证，我们首次发现miR-21具有促进间充质干细胞血管及骨向分化作用。鉴于此，本项目拟构建大鼠及山羊ORN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-21介导的血管化组织工程骨对ORN生物功能重建作用及其信号转导通路的调控。

颅颌面标准骨缺损 (CSD) 的修复重建是临床医学面临的一大挑战，研究证实基于组织工程的骨再生技术是 CSD 修复的新途径。在本课题的资助下：(1) 体外实验结果表明miR-21 具有促进 MSCs 血管向分化作用，首次利用 miR-21 介导的人脐带血干细胞治疗裸鼠下肢缺血，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-21 作用于靶基因 CHIP 激活 HIF-1α 的活性，进而促进了血管的再生及分化。该部分研究结果发表在国际著名的干细胞杂志上 (Stem Cells. 2016 Apr;34(4):924-34.)。(2) 课题组成功构建了大鼠下肢 ORN 模型，这为 ORN 研究提供了研究基础，为此，我们申请了 1 项专利 (大鼠下肢放射性骨坏死模型构建装置)，发表了一篇中文核心期刊 论文 (上海口腔医学, 2016, 25(5):513-517 )。(3) 由于山羊下颌骨 ORN 模型构建不理想，我们对课题进行了调整，进行了大鼠颅骨及犬下颌骨标准化骨缺损进行了修复研究。研究结果表明：miR-21 具有促进 BMSCs 骨向分化的作用，并明确了 miR-21 作用于靶基因 PI3K 及 Akt 激活 HIF-1α 通路，进而促进 BMSCs 骨向分化。体内应用 miR-21 介导的 BMSCs 构建组织工程复合体，进行标准骨缺损修复，结果表明其可以有效修复大鼠颅骨及犬下颌骨标准骨缺损 (under review)。通过本课题的研究，我们明确了 miR-21 为促进 MSCs 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-21 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。