GARP-TGFβ1蛋白复合体在骨髓间充质干细胞抑制炎性牙槽骨吸收中的作用研究

Alveolar bone absorption is a common result of chronic inflammatory cell infiltration in periodontitis. The alveolar bone regeneration after periodontitis treatment remains unsatisfactory in current clinical practice. Bone marrow mesenchymal stem cells (BMMSCs), which possess immune-regulatory properties, might have a promising therapeutic effect on the inflammatory bone absorption of periodontitis. Our previous studies showed that (1) When co-cultured with CD4+T lymphocyte, BMMSCs with highly expressed Transforming Growth Factor beta 1 (TGF-β1) can induce the differentiation of regulatory T cells (Tregs) and inhibit the expression of inflammatory cytokines released by other pro-inflammatory T cell subtypes, thus prevent themselves from apoptosis. (2) In the rat periodontitis model, the inflammatory alveolar bone absorption can be suppressed by application of rats’ recombined TGF-β1 protein gel in their gingival sulcus. Therefore, TGF-β1 could be the critical factor in modulating alveolar bone regeneration. TGF-β1 is produced as a latent form, which could prompt to be activated when binding with a transmembrane protein glycoprotein A repetitions predominant (GARP) on BMMSCs. The relocalization of intracellular GARP to the surface of BMMSCs can be conferred to its polypeptide-folding reaction when it binds to its molecular chaperone, which might play very important role in controlling the activation of TGF- β1. Based on these findings, we suggest that (1) The BMMSCs possibly exert their immune-regulatory effect through the GARP-TGFβ1 complex. (2)The up-regulation of membrane locating GARP might amplify their immunosuppressive capability. We would utilize various kinds of techniques such as bioinformatics analysis, protein co-immunoprecipitation and RNA interferences to test our hypothesis. By decoding the detailed mechanism of how active TGF-β1 is provide by BMMSCs to suppress the inflammation, we would like to provide a new perspective on treating inflammatory bone absorption in the clinical context.

牙周炎骨缺损修复手段尚有局限性，修饰后的骨髓间充质干细胞（BMMSCs）具有应用前景。我们前期研究发现：高表达转化生长因子β1（TGF-β1）的BMMSCs细胞凋亡减少并可诱导调节性T细胞的分化、抑制炎症因子的分泌；局部注射TGF-β1可减缓牙周炎牙槽骨吸收，提示了高表达TGF-β1的BMMSCs在牙周炎骨缺损修复中的作用。BMMSCs胞膜上的富含亮氨酸重复序列蛋白（GARP）是TGF-β1的受体，其从胞内转运至胞膜并与TGF-β1结合后使其活化。据此我们提出假说：BMMSCs细胞膜上的GARP-TGFβ1蛋白复合体是其发挥免疫调节作用的关键；上调GARP在胞膜上的表达将增加GARP-TGFβ1蛋白复合体形成进而增强BMMSCs的功能。我们拟从分子、细胞、组织和动物水平来验证我们的假说，并探讨GARP细胞内跨膜转运的调控机制，为炎性骨吸收的治疗提供新的思路。

骨髓间充质干细胞（Bone Marrow Mesenchymal Stem Cells, BMMSCs）广泛存在于血液、骨、脂肪等组织中，且具有多项分化潜能，可以在特定条件下分化为骨细胞、软骨细胞和脂肪细胞。因此，BMMSCs可以作为组织工程引导骨再生的理想载体。基因修饰的BMMSCs在修复牙周炎骨缺损领域具有良好的应用前景。前期研究发现，高表达TGF-β1的BMMSCs可以修复牙周炎骨缺损，而BMMSCs胞膜上的亮氨酸重复序列蛋白（GARP）可以与TGF-β1结合并参与TGF-β1的活化。为了研究此调控机制，我们通过基因转染技术获得了BMMSCsGARP-稳转株，并在基因和蛋白层面验证其稳定低表达。通过体外培养BMMSCsGARP-，我们发现GARP的低表达，可以下调活化TGF-β1的表达，进而下调BMMSCs成骨相关蛋白ALP、Runx2和OPN的表达，这提示了GARP在骨改建过程中发挥了重要作用。为了验证该结论，我们通过建立兔颅骨缺损模型开展体内实验。在组织工程中，安全可降解的生物学支架是颅骨缺损修复模型的关键因素，因此我们选择可注射型蛋白水凝胶作为支架材料进行研究。在本项目的资助下，我们对该新型材料开展了充分的研究，该支架材料以银离子（Ag+）为交联剂，构建网状交联结构，体外抑菌实验表明，随着支架材料的降解，Ag+缓慢释放发挥抗菌效果，保证成骨的有利微环境。该蛋白水凝胶包含支持细胞粘附和生长的有效基质，具备良好的生物相容性。动物颅骨缺损模型进一步验证了蛋白水凝胶具备良好的引导骨再生的生物学性能，是骨组织工程的理想支架材料。本课题为进一步构建基因修饰的BMMSCs负载到可注射型蛋白水凝胶支架的研究提供了理论基础和实验依据。