HSPA1A修饰BMSCs复合CD31（+）细胞预血管化的生物膜在治疗大段骨缺损的作用及机制研究

It is urgent to find a novel approach to the treatment of segmental bone defect. Rapid vascularization is essential to the successful treatment. However，in current bone tissue engineering, there are no ideal seed cells to succeed in osteogenesis and vascularization simultaneously. Meanwhile, the survival rate of cells implantation is very low. Thus, those decrease the therapeutic effects of stem cells implantation severely. Our previous study found that the expression of heat shock protein family A member 1A (HSPA1A) not only increase the survival rate of bone marrow derived stem cells transplantation, but enhanced the ablilites of osteogenesis and paracrine, through VEGF/PI3K/AKT signaling pathway, thereby promoting the homing of endothelial cells and stem cells. Moreover, CD31（+） cells in peripheral blood contain highly angiogenic and vasculogenic cells. More importantly, they are easy to obtain due to large in amount. Therefore, we put forward a hypothesis that a multilayer sheet of HSPA1A-modified BMSCs, which is pre-vascularization by CD31（+） cells transplantation, could improve the healing of segmental bone defect. Of note, the biological membrane would simulate Masquelet technique. Moreover, we struggle to investigate its potential mechansim as well, thereby providing a new insight for the treatment of segmental bone defect in tissue engineering.

大段骨缺损的治疗亟需新的治疗手段，其中缺损区域的快速血管化是治疗成功的关键之一。而现今组织工程种子细胞难以兼顾骨形成和血管化，同时植入的生存率也很低。这样严重影响了细胞治疗的效果。我们前期的研究发现，热休克蛋白A1A基因（HSPA1A）的表达不仅增强骨髓间充质干细胞（BMSCs）植入时的存活能力，同时HSPA1A通过VEGF/PI3K/AKT通路增强其成骨分化能力并增强其旁分泌功能（VEGF、SDF-1a），促进细胞的迁移。外周血CD31（+）细胞具有较强的成血管能力，且含量多、易提取。因此，本课题组将应用相关实验技术探索HSPA1A修饰的BMSCs治疗骨缺损、加速血管化的分子机制，同时将HSPA1A修饰的BMSCs的细胞膜片结合CD31（+）细胞，构建预血管化的多层生物膜片，模拟Masquelet诱导膜成骨技术，修复大段骨缺损，从而为骨血管化组织工程修复大段骨缺损提供新的思路。

HSPA1A编码同源热休克蛋白70，在多种细胞中发挥重要作用代谢途径。在本项目中，我们主要探讨了HSPA1A对骨髓间充质干细胞(mesenchymal stem cells, BMSCs)成骨分化的影响，未分化和分化的bmsc的表达水平比较。大鼠骨髓间充质干细胞中HSPA1A过表达增加了成骨细胞特异性基因碱性磷酸酶活性和矿物沉积在体外。此外,它调节β-catenin和下调DKK1和SOST。HSPA1A促进骨形成Wnt/β-catenin抑制剂可部分挽救过表达。此外，使用大鼠胫骨骨折模型中，hspa1a过表达的骨髓间充质干细胞片促进骨折愈合影像学及组织学分析。综上所述，这些发现表明HSPA1A过表达部分通过Wnt/β-catenin增强骨髓间充质干细胞的成骨分化。.据我们所知，这是首次研究HSPA1A对间充质干细胞成骨分化的影响。我们发现，rBMSCs成骨过程中内源性HSPA1A表达上调。此外，根据以往的研究结果，HSPA1A在新的骨愈合区域高表达。因此，我们想使用HSPA1A过表达策略来加速MSCs的成骨。我们发现HSPA1A过表达部分通过Wnt/β-catenin信号通路促进rBMSCs的成骨分化体外途径。此外，一层过表达HSPA1A的rBMSCs可促进骨折愈合大鼠骨折模型。这些结果表明HSPA1A过表达可促进成骨分化rBMSCs，至少部分通过激活Wnt/β-catenin信号通路。