Notch1依赖的KLF2+间充质干细胞促进骨的功能性血管网再生的作用及机制研究

“功能性血管网”的构建是目前组织工程骨临床转化的主要障碍和难点。申请人通过前期研究发现KLF2+干性维持的MSCs可能促进血管内皮细胞（ECs）功能性血管网生成（Stem Cells，2020），且与Notch1密切相关，但其分子机制尚未明了。因此提出科学假说：KLF2+MSCs可能通过分泌VEGF并激活Notch1通路，诱导自身向周细胞转化，从而促进ECs功能性血管形成。本项目拟运用KLF2过表达，明确KLF2+MSCs促进ECs功能性血管网形成；以MSCs周细胞转化为切入点，揭示KLF2+MSCs通过激活Notch1通路，诱导自身向周细胞转化，促进ECs功能性血管网形成的机制。最后运用光控细胞薄层技术构建功能性血管网，动物体内验证。本研究从“干性维持”的新视角出发，探索KLF2+MSCs在“功能性血管网”构建中的重要作用，为开发预血管化的植入物修复口腔颌面部骨缺损提供必要的理论依据。

The reconstruction of large bone defects using bone tissue engineering remains a major challenge in clinic due to the inadequate vascularization of implanted grafs. Mesenchymal stem cells (MSCs) co-cultured with endothelial cells (ECs) to generate prevascularized constructs in vitro have attracted the most attention in strategies that seek to achieve vascularized bone. In our previous study, we have developed a kind of culture medium (medium 1) for the stemness maintenance of MSCs and identified Klf2 as a crucial transcription factor. Then, different stemness stage of MSCs were generated using medium 1 and KLF2 knock down to explore the interaction between stemness maintained MSCs and ECs. The results indicated that MSCs maintained in stemness may promote the angiogenesis of ECs and osteogenesis itself later in co-cultures. However, the intrinsic mechanism underling this process remain poorly understood. Using KLF2 over expression, the aim of our present study was to verify the effect of angiogenesis and osteogenesis of KLF2+ MSCs during MSCs/ECs co-culture and identify the possible mechanism through the activation of Notch1 pathway in regulating MSCs to differentiate into pericytes. This work should shed light on the fundamental understanding of stemness directed synergistic strategy between MSCs and ECs, and will have a profound impact on further strategies of preferring KLF2+ stemness maintained MSCs in vascularized bone regeneration.