LncRNA H19在羊膜间充质干细胞促进种植体周围骨再生中的作用机制研究

Our previous studies showed that the amniotic mesenchymal stem cells (AMSCs) can promote bone regeneration to repair bone defects around implants through their ability of inducing angiogenesis, however, the related mechanism is still unclear. According to gene microarray tests, the expression of LncRNA H19 and miR-675 encoded by H19 has been significantly increased in the process of angiogenesis caused by AMSCs. Posterior to knocking down the level of H19, less angiogenesis and lower expression of miR-675 has been examined, while the level of Smad7 (a repressor of angiogenesis involved TGF-β/Smad pathway) has been dramatic increased. Bioinformatics analysis manifested that binding sites for miR-675 may exist in Smad7 mRNA 3'UTR region. H19 therefore may inhibit the expression of Smad 7 by the combination of its encoding product miR-675 and Smad7 mRNA 3 'UTR region, which would activate TGF-β1 pathway and accelerate angiogenesis. Our study will demonstrate the hypothesis above according to molecule biology, cytology, and animal experiments, and the results may explain the mechanism of H19 related to new bone formation facilitated by AMSCs, which provide basis for treatment of bone defects around implants.

我们研究发现，羊膜间充质干细胞（AMSCs）能够通过促进血管新生，进而促进骨再生，修复种植体周围骨缺损，但其作用机制不明确。我们经基因芯片检测发现：AMSCs在促进血管新生过程中LncRNA H19、H19编码生成的miR-675表达明显增高。干扰H19的表达后可见：血管新生明显减少，miR-675表达水平降低，与血管新生相关的重要信号通路TGF-β/Smad信号通路抑制因子Smad7的表达水平明显增高。生物信息学分析发现：Smad7 mRNA的3’UTR区存在miR-675可能的结合位点。由此提出：H19通过编码生成的miR-675与Smad7 mRNA的3’UTR区结合，抑制Smad7表达，激活TGF-β1信号通路，使血管新生增加。本研究拟从分子、细胞及动物体内3个层面验证此假说，这将对LncRNA在AMSCs增殖、分化中的作用机制研究具有重要意义，为治疗种植体周围骨缺损提供实验依据。

本课题组通过构建人羊膜间充质干细胞（HAMSCs）和人脐静脉血管内皮细胞（HUVEC）三维共培养体系，发现H19在HAMSCs促进HUVEC成管表型中起着重要作用，深入研究发现HAMSCs内H19可通过结合EZH2调控VASH1 DNA甲基化从而促进VASH1分泌，进而调控HUVEC血管新生。进一步体内初步建立兔上颌窦区种植体周围骨缺损模型发现：HAMSCs联合Bio-Oss较空白组、单纯使用HAMSCs组、单纯使用Bio-Oss组，可显著促进兔上颌窦区种植体周围骨缺损的成血管及成骨。继而在兔颅骨缺损模型中的研究发现HAMSCs可通过抑制炎症、促进血管新生及促进成骨进而促进兔颅骨缺损的修复。在此基础上，我们探索了其他类型的非编码RNA是否在此过程中发挥着重要作用，我们发现HAMSCs上清可显著促进HUVEC内环状RNA circ-100290表达，进一步研究发现其可通过miR-449a/eNOS轴及miR-449a/VEGFA轴促进HUVEC血管生成。基于以上结果，本课题组发表5篇英文论著。