miR-210双重调控BMSCs构建血管化组织工程骨复合体修复CSD及靶基因调控机制的研究

This project aimed at the clinical issues that demanding urgent solution to effectively repair and reconstruct critical bone defect (CSD), to actively explore the effect and mechanism of growth factors with the dual role on repairing CSD by gene-enhanced technique. It is reported that miR-210 is a downstream target gene of HIF-1 alpha, which play an important role in vascular development and generation. The researchers found that implanting BMSCs over expression of miR-210 may be effective in the treatment of lower limb ischemia, however there has not been reported that whether it can promote bone formation and regeneration so far. We speculate that miR-210 can promote the osteogenesis and vascularization.differentiation of BMSCs. Besides, we successfully have constructed tissue engineering composite using HAP-chitosan microspheres. This hypothesis has been verified in our pre experiment: miR-210 holds the dual role in promoting BMSCs into vascular and bone differentiation, Sclerostin is the target gene of miR-210. In view of this, the project intends to construct the rat and dog animal model of CSD, apply the lentiviral vector transduction, gene chip detection, vascularized tissue engineering bone construction and dental implant implantation technique, verify our hypothesis at 4 aspects that is mRNA, protein, small animals, large animals, and clarify the regulation mechanism of miR-210 on constructing vascularized tissue engineering composite to repair CSD and its target genes.

本项目瞄准临床上大量标准骨缺损(CSD)亟待有效修复与重建这一临床课题，积极探索双重作用生长因子在利用基因增强组织工程技术修复CSD中的作用及机制。文献报道miR-210是HIF-1α的下游靶基因，其在血管发育和再生中发挥重要作用。研究发现BMSCs过表达miR-210可有效治疗下肢缺血，然而是否具有促进骨再生作用，迄今国内外尚未报道，本课题组预实验发现miR-210具有促进BMSCs血管及骨向分化双重作用，且明确了Sclerostin为其骨向分化作用的靶基因。另外，我们利用羟基磷灰石/壳聚糖中空微球生物支架成功构建了组织工程复合体。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、血管化组织工程复合体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复合体对CSD生物功能重建作用及其调控机制。

标准骨缺损（CSD）修复重建是临床亟待解决的难题。本项目在前期研究基础上，利用大鼠颅骨及犬下颌骨 CSD 动物模型，应用慢病毒载体转染、基因芯片、血管化组织工程复合体构建等技术，从 mRNA-蛋白-小动物-大动物 4 个 层次，多角度观察 miR-210 介导的 BMSCs 在 CSD 修复中的作用和相关机制。结果发现：（1）miR-210 下调靶基因Sclerostin，促进成骨相关蛋白的表达，从而促进BMSC骨向分化。（2）miR-210通过显著上调HIF-1α、VEGF、BMP-2、OPN、OCN和Runx2等成血管及成骨相关基因从而促进成骨。（3）成功构建了大鼠颅骨及犬下颌骨 CSD 模型, 并初步证明miR-210/BMSCs 构建的组织工程骨可以有效进行大鼠颅骨及犬下颌骨CSD的修复。本项目的研究为将来 miR-210/BMSCs 构建的组织工程骨应用于临床的 CSD 修复提供理论和实验依据。