炎性微环境下TGF-β1/Treg相关细胞因子介导的免疫调控在骨髓间充质干细胞骨向分化中作用机制研究

Pathological alveolar bone resorption is the hallmark feature of periodontitis. Eventually lead to teeth loose or falling off. Recently, stem cells has used to repair the alveolar bone defect and restoring the physiological function of the natural tooth. Previous studies shown that mesenchymal stem cells (MSC) promote the formation of regulatory T cell (Treg), which is mediated directly by transforming growth factor beta 1 (TFG-β1). However, the mechanism of TGF-β1/Treg-related cytokines mediated immune regulation on osteogenic differentiation of bone marrow mesenchymal stem cells under inflammatory microenviroments is not fully identified. Bone marrow mesenchymal stem cells (BMMSCs ) displayed anti-inflammatory capacities in our previous studies. And the expression of TGF-β1 and IL-10 upregulated. This study intends to verify that the TGF-β1 and Treg-related cytokines play an important role in the regulation of BMMSCs osteogenic differentiation. The experiment consists of three parts as follows: (1) neutralizing antibodies against the TGF-β1 receptor and the inhibitor of the TGF-β1 receptor; (2) overexpression or siRNA of TGF-β1 and Foxp3 in BMMSCs transduced with lentiviral vectors; and (3) observation of the ability of BMMSCs in restoring alveolar bone defects in vivo based on in vitro regulation. These researches provide a theoretical basis for the study of bone regeneration based on stem cells and tissue engineering.

牙槽骨病理性吸收是牙周炎的标志性特征，最终导致牙齿松动甚至脱落。干细胞修复缺损牙槽骨打破了以往治疗的瓶颈。间充质干细胞（MSCs）通过分泌转化生长因子β1（TGF-β1）诱导调节性T细胞（Treg）形成在炎症和免疫应答中发挥重要作用，然而TGF-β1和Treg相关细胞因子介导的免疫调控对MSCs骨向分化作用机制尚不清楚。我们前期研究证实炎症环境下骨髓间充质干细胞（BMMSCs）抗炎能力较强，TGF-β1和IL-10表达增高，本项目拟通过①中和抗体和抑制剂实验阻断TGF-β1受体；②构建TGF-β1和转录因子Foxp3过表达及siRNA的慢病毒载体，转染BMMSCs；③体外调控基础上观察BMMSCs体内修复牙槽骨缺损；证实炎性微环境下TGF-β1和Treg相关细胞因子介导的免疫调节对BMMSCs骨向分化有重要的调控作用，为建立基于干细胞和组织工程技术的骨组织再生研究提供理论依据。

牙槽骨的病理性吸收最终导致牙齿松动甚至脱落。牙周膜干细胞 (PDLSCs) 在一定条件下可以分化成成骨/成牙骨质细胞、与生物材料构建的复合物能够治疗牙周组织缺损。然而其来源和数量有限，与PDLSCs相比，骨髓间充质干细胞 (BMMSCs) 取材方便、来源广泛，易在体外培养、扩增和诱导。以往研究证实BMMSCs在修复牙槽骨缺损区中成骨能力高于PDLSCs。我们前期实验结果也证实相同刺激条件下BMMSCs成骨能力高于PDLSCs。最近研究证实MSCs能够发挥抗炎功能的一个重要机制是其能分泌可溶性因子，这些可溶性因子使MSCs与免疫细胞相互作用，诱导Treg细胞形成，通过调节局部微环境、减少炎症因子分泌发挥其抗炎和免疫抑制作用。然而 BMMSCs与Treg细胞之间的免疫调控机制目前尚不清楚。我们前期实验将 Treg细胞与BMMSCs在炎性环境下共培养，结果表明Treg细胞参与并促进BMMSCs的分化；进一步提取 Treg 细胞的外泌体，与BMMSC共培养证实 Treg 细胞外泌体能够促进 BMMSCs骨向分化标志物和基因的表达；染色实验及PCR、Western Blot证实Treg来源外泌体能进入BMMSC内，通过调控BMMSC内POSTN蛋白的表达，从而促进了BMMSCs的骨向分化；通过Starbase, miRWalk和TargetScan数据库及荧光素酶报告基因和 Western blot 技术证实 miR-154负性调控了POSTN的表达；应用生物信息学技术（LncRNA数据库分析）文献回顾及实验证实Treg细胞外泌体内lncRNA H19与BMMSCs内miR-154-5p相结合，通过调控POSTN蛋白的表达在BMMSCs骨向分化中发挥重要作用。因此，应用Treg细胞外泌体作为免疫调控剂将为牙周炎的骨组织再生提供新的思路和方法。