功能性微血管化自体生物骨动态追踪及基因筛选

Necrosis and absorption of bone grafts caused by rejection and insufficient blood supply after bone transplantation, which induced failure of bone defect reconstruction and serious organism damage caused by sphacelus. The key point for clinic treatment of bone defect was to research and develop an engineering bone with good histocompatibility, microcirculation, no rejection and can be regulated by organism. The prophase research of our research group indicated that the cell proliferation and osteogenesis of vascular endothelial cells (VECs) and bone marrow stem cells (BMSCs) co-culture system were significantly better than single BMSCs, and there is BMP-2/Runx2 pathway in co-culture system, but BMP-2 was not the only affectoi of upstream pathway of Runx2. In this research, autologous EPCs with brilliant neovascularization capability isolated from peripheral blood and BMSCs with secretion capability of cytokine which can promoting lumen of blood vessel formation isolated from autologous bone marrow were co-cultured together, and seeded onto partially deproteinised biologic bone. The gene expression of co-culture system were screened with genechip, and downstream pathway of co-culture system are investigated. In vitro and vivo, the properties of engineering biological bone such as cell proliferation, immigration， lumen formation, the vascularization of EPCs，the osteogenesis of BMSCs, the osteogenesis, microcirculation formation, bone repair and rejection of engineering bone were dynamic monitored. The object of this research is to provide the systematized technology for constructing an autologous engineering biological bone with good histocompatibility， microvascularization and microcirculation, which can be fit for clinic application.

骨移植术后因排异反应、血供不良，导致移植骨坏死、吸收，使骨缺损修复失败，且坏死物能对机体造成严重损伤。研发组织相容性好、血液供应充足及受机体调控的组织工程骨，是进入临床应用的关键。课题组前期研究表明，血管内皮细胞与骨髓间充质干细胞联合培养体系在细胞增殖、成骨方面均有明显优势；联合培养体系中存在BMP-2/Runx2 通路，但BMP-2 非Runx2 通路的唯一影响因子。本研究采用来源于自体具有优异新生血管能力的外周血内皮祖细胞（EPCs）及可分泌促管腔形成细胞因子的骨髓间充质干细胞（BMSCs）构建联合培养体系，复合脱蛋白生物骨（PDPBB）构建组织工程骨；基因芯片检测联合培养体系基因表达变化，筛选下游通路；对体外微血管管腔形成、成骨能力、活体内微循环血液灌流、骨缺损修复及排异情况等生物骨性能进行动态追踪。为构建无排异反应、微循环良好的自体组织工程生物骨应用于临床提供系统化技术支持。

近年来再生医学快速发展，建立组织工程骨修复骨缺损成为研究重点。修复骨缺损最重要的环节是骨愈合过程中迅速建立血管化从而使组织工程骨处于代谢和生长的微环境中。血管内皮祖细胞（Endothelial Progenitor Cells，EPCs）能够形成血管网状结构, 促进管腔的形成和微循环通路的建立。本实验通过血管内皮祖细胞与骨髓间充质干细胞复合脱蛋白生物骨构建复合细胞-生物骨，从EPCs对BMSCs的增殖、分化、和归巢三方面探索BMSCs修复骨缺损机制。应用 Transwell 体外共培养的方法观察EPCs对 BMSCs 的体外增殖和诱导分化的作用。应用稳定表达eGFP的BMSCs注入活体内，观察外源性 BMSCs 在体内骨缺损部位归巢情况；检测基质细胞衍生因子和受体蛋白CXCR4以及单核细胞趋化蛋白1和其受体CCR2向活体骨缺损处归巢的关键作用，为骨缺损的干细胞治疗提供新的实验依据和理论支撑。主要发现和结论： 采用贴壁法分离、纯化兔BMSCs和EPCs的方法有效；EPCs：BMSCs 为10:1为最佳比例复合培养组；复合细胞组中的BMSCs体现出更强的细胞增殖活性、更有潜力的成骨分化能力；复合细胞培养组具有更强的促进外源性BMSCs向骨缺损处归巢的作用；SDF-1/CXCR4和MCP-1/CCR2两条趋化轴与EPCs促进BMSCs向骨缺损部位趋化运动密切相关，其中以SDF-1/CXCR4轴联系更明显；EPCs和BMSCs与课题组制备的脱蛋白生物骨复合后可更有效、更快速进行骨缺损修复；制作的mRNA芯片样本，可有效筛选复合培养组和对照组差异表达的基因。结合课题组之前构建联合细胞的体内研究，全面阐释EPCs与BMSCs复合细胞体系在促进组织工程骨微血管化、成骨和归巢的强大优势，为加速组织工程骨的构筑时效和促进骨组织工程的临床转化奠定理论基础。