骨膜蛋白介导CTLA4Ig修饰的MSCs在异基因移植中成骨的作用及机制研究

As a key factor of bone tissue microenvironment, periostin plays a vital role in promoting the bone defect repairing. In our previously study, we confirmed that the CTLA4 modified mesenchymal stem cells (MSCs) could form new bone tissues successfully in allotransplantation, but the most cells of new bone formation were from the host. Besides, we also found the periostin expression were significantly higher in CTLA4 modified MSCs. Periostin can bind to αvβ3-integrin to mediate the interaction between cell and extracellular matrix. Moreover, the periostin also can induce the osteogeic differentiation of MSCs directly and recruit MSCs through the concentration gradient in blood, however, the detail mechanism remains to be revealed. From the above, we make a hypothesis that CTLA4 modified MSCs may recruit the host MSCs through the binding between periostin and αvβ3-integrin to promote the bone defect repairing in allotransplantation. Based on works we had done before, we plan to use periostin-siRNA, Transwell migration assays and orthotopic transplantation of tissue-engineered bone in RFP mouse skull-defect model to verify this hypothesis. This reasearch will clarify the osteogenesis mechanism of CTLA4 modified MSCs in allotransplantation, and provide a thecretical basis for its clinical application.

骨膜蛋白作为骨组织微环境的关键成分，在促进骨缺损的修复中发挥着重要作用。我们前期研究发现，免疫负调分子CTLA4修饰的间充质干细胞（MSCs）在异基因移植时能够有效成骨，且新生骨组织细胞多来源于宿主；同时在CTLA4修饰的MSCs中，骨膜蛋白的表达显著增高。骨膜蛋白可通过结合整合素αvβ3来介导细胞与胞外基质的相互作用，在诱导MSCs成骨分化的同时能通过血液中的浓度差来募集MSCs，但具体机制有待探索。由此我们推测：在异基因移植中，CTLA4修饰的MSCs通过骨膜蛋白结合整合素αvβ3来增强对宿主MSCs的募集，从而促进骨缺损的修复。本研究拟在前期工作基础上，通过利用骨膜蛋白siRNA基因沉默技术、Transwell趋化模型和组织工程骨原位移植荧光小鼠颅骨缺损模型来共同验证该假说，进一步阐明CTLA4修饰的MSCs在异基因移植中促进骨缺损修复的具体机制，为其今后的临床应用转化提供理论依据。

大段骨缺损在骨科临床工作中十分常见，而通过组织工程骨（TEB）策略修复大段骨缺损已被证实具有良好的疗效。在前期工作中，我们将细胞毒性T淋巴细胞相关抗原4免疫球蛋白（CTLA4）基因导入人骨髓间充质干细胞（MSCs）中，构建出表达CTLA4的MSCs，发现其在异体移植过程中能够有效成骨，但具体机制不明。随后我们发现，在异基因移植的TEB-MSCs-CTLA4组中，骨膜蛋白（POSTN）的表达显著高于对照组。结合相关文献查询，我们提出假设：在异基因移植中，CTLA4修饰的MSCs可通过分泌POSTN来增强对宿主MSCs的募集作用，从而促进骨缺损的修复。为验证该假设，本次研究我们利用腺病毒载体技术，成功构建出了抑制POSTN表达的MSCs-CTLA4，通过相关分子生物学技术，不仅从细胞趋化方面证实了POSTN的存在可以有效加强MSCs的趋化效应，还从分子机制上进一步揭示了POSTN可通过结合MSCs上的αvβ3-integrin，进而诱发其下游FAK、AKT及ERK的磷酸化水平，以此促进MSCs分泌基质金属蛋白酶-2(MMP-2)，启动MSCs的粘附和趋化效益，募集宿主MSCs参与成骨修复。本研究阐明了CTLA4修饰的MSCs在异基因移植中通过分泌POSTN，进一步结合αvβ3-integrin来激活下游FAK/Paxillin通路，从而募集宿主MSCs参与骨缺损修复的具体机制，为今后研发TEB的理想种子细胞提供了新的理论依据和研究方向。