基于miRNA转染的血管化BMSC膜片的构建、血管化与骨再生能力及其机制研究

Currently, the cell sheet engineering has attracted increasing attention in the field of regenerative medicine. In particular, the bone marrow mesenchymal stem cell (BMSC) based cell sheet engineering has already been applied for the repair and regeneration of bone tissue defects. However, the main hurdle of current bone tissue engineering is how to obtain sufficient blood supply to the bone tissue graft. One good strategy for overcoming this obstacle could be the development of prevascularized BMSC sheet based tissue constructs before transplantation. Based on our previous study, we have successfully delivered antimiR-138 to the BMSC sheets by using the non-viral way. To our knowledge, this is the first attempt to use the non-viral approach to deliver oligonucleotides to the cell sheets. We also find that that the antimiR-138 delivery significantly enhances the osteogenesis of BMSC sheets. Moreover, considering the interactions of miR-126 with the vascular endothelial cells (EC) play a crucial role in the development of angiogenesis and the maintenance of vascular integrity, we plan to co-culture the antimiR-138 delivered BMSC sheets with the miR-126 overexpressed endothelial cells to fabricate the prevascularized cell sheets containing the capillary-like networks in vitro. A systemic study will be conducted on the osteogenesis and angiogenesis effects of the miRNA-MSC/EC sheets. Then, we will try to uncover the underlying molecular mechanism for the fast vascularization of the miRNA-MSC/EC sheets. Our present study aims to successfully construct the prevascularized miRNA-MSC/EC sheets, which may provide new idea for the development of vascularized tissue engineered bone. Meanwhile, the results also provide more evidences to support the application of miRNA delivery technology in the regenerative medicine.

细胞膜片技术是现代再生医学的一个研究热点，骨髓间充质干细胞（BMSC）膜片已广泛用于骨缺损的修复与再生。血管化一直是制约骨组织再生效果的瓶颈，如何实现膜片的快速血管化以达到理想的基于BMSC膜片的骨再生是非常有意义的一个科学问题。本课题组前期在国际上首次采用非病毒转染技术将antimiR-138转入BMSC膜片后发现其可显著提高BMSC膜片的成骨能力，同时考虑到miR-126对血管内皮细胞在血管发生中的重要促进作用，我们提出将antimiR-138修饰的BMSC膜片与miR-126修饰的血管内皮细胞连续共培养来构建预血管化BMSC膜片，体内外实验对其骨再生和血管生成作用进行系统评估，并探讨其快速血管化的分子机制。本研究有望成功构建血管化BMSC膜片，为骨组织工程血管化提供新的思路，亦为miRNA在再生医学中的应用提供更多的证据，为miR-126促血管化的分子机制提供更深入的理解。

细胞膜片技术是现代再生医学的一个研究热点，骨髓间充质干细胞（BMSC）膜片已广泛用于组织缺损的修复与再生。血管化一直是制约大面积组织再生效果的瓶颈，如何实现膜片的血管化以达到基于BMSC膜片更好的组织再生是非常有意义的科学问题。干细胞潜在的自分泌和旁分泌多种细胞因子和免疫调节功能可能参与调节正常血管的生成与重塑。研究目的：1.构建预血管化的EC-BMSC复合膜片；2.探索BMSC调控膜片血管化的旁分泌机制；3. 通过体内实验综合评估血管化膜片的功能。研究方法：1.通过EC与BMSC膜片共培养技术在体外自组装形成预血管化的膜片，采用免疫荧光染色观察不同配比的共培养培养基对EC-BMSC 膜片血管网形成及BMSC 膜片干性的影响。2.采用蛋白芯片技术分析共培养上清中相关分泌因子表达的差异，采用 Western blot 分析膜片成血管相关蛋白表达的差异。3.通过体内静脉 Evans 蓝灌注实验，组织切片的免疫荧光染色观察膜片血管与宿主血管的吻合。研究结果：1.成功通过共培养技术在体外自组装形成预血管化的EC-BMSC复合膜片，并证明1：1EBM2/SIM组的成血管效果最好。2. 1：1EBM2/SIM组自分泌的VEGF、Ang-1、Ang-2、MMP-9、MMP-2等血管相关蛋白与其血管生长的需求更匹配，且通过激活Ang-1、Ang-2/Tie2信号通路促进了血管的生成。3、体内免疫荧光染色提示1：1EBM2/SIM组血管壁发育最成熟，且膜片血管与宿主血管形成良好吻合。结论：本研究成功构建血管化EC-BMSC复合膜片，为组织工程血管化提供新思路，为干细胞调节血管生成的旁分泌机制提供更深入的理解。