双基因活性真皮支架介导VEGF和Angiopoietin-1共表达及序贯性调控血管化进程的研究

The angiogenesis process induced by dermal substitutes, including vascular lumen formation and vascular maturation is precisely regulated by various bioactive factors in the in vivo microenvironment. As known, there are the synergetic effects between vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) to sequentially regulate the process of angiogenesis. VEGF, one of the strongest angiogenic factors, can stimulate endothelial cells to proliferate, migrate and further to form vascular lumens, while Ang-1 plays an important role in vascular maturation by inducing pericytes to wrap the vascular lumens. How to realize the effective delivery and sustained impact of these angiogenic factors is crucial to promote the angiogenesis process of dermal substitutes especially at the early stage after transplantation. Previous studies indicate that gene-activated dermal scaffolds provide a new pattern to effectively deliver and controllably release angiogenic factors. We hypothesize that double gene-activated dermal scaffolds (DGADSs) can be constructed by loading the gene vector coated plasmid DNA of VEGF and Ang-1. When transplanted into the skin wounds, DGADSs have the potential to realize the efficient transfection of desired genes, and continuously release VEGF and Ang-1 to sequentially regulate the process of angiogenesis. In order to prove the hypothesis described above, the full-thickness skin defect model, as well as the cell culture model will be employed to optimize the construction methods of DGADSs, and further to investigate the effectiveness of DGADSs in sequentially regulate the vascular lumen formation and vascular maturation. The results will provide a new solution for promoting angiogenesis, and also provide more theoretical bases for the development of novel dermal substitutes.

真皮替代物体内的血管化进程，从血管新生到成熟受到多种生物活性因子的精确调控。如VEGF和Angiopoietin-1（Ang-1）具有重要的协同作用，前者促进内皮细胞的迁移、增殖和初级管腔形成，而后者作用于血管周细胞并促进血管成熟。如何实现这些促血管活性因子的高效传递和持续作用是解决真皮替代物早期血管化不足的关键。前期研究表明，基因活性真皮支架是实现促血管活性因子持续表达的有效手段。我们假设将VEGF和Ang-1的质粒DNA以一定方式整合到真皮支架上构建双基因活性真皮支架，可实现两种基因在创面局部的高效转染并表达，进而更加精准地实现对血管新生与成熟的序贯性调控。为证明上述假设，本研究拟采用体内移植模型和体外细胞培养模型优化双基因活性真皮支架的构建并验证其有效性，进而揭示其序贯性调控血管化进程的作用机理，为真皮支架早期血管化问题提供新的解决方案，为新型真皮替代物的开发提供依据。

项目背景：各种急慢性致伤因素导致的皮肤组织缺损在临床上十分常见。真皮支架的出现为临床上深度皮肤缺损的治疗提供了一条较为理想的途径。目前，真皮替代物早期血管化不足的问题亟待解决。真皮替代物体内的血管化进程，从血管新生到成熟受到多种生物活性因子的精确调控。VEGF和Angiopoietin-1（Ang-1）具有重要的协同作用，前者促进内皮细胞的迁移、增殖和初级管腔形成，而后者作用于血管周细胞并促进血管成熟。如何实现这些促血管活性因子的高效传递和持续作用是解决真皮替代物早期血管化不足的关键。前期研究表明，基因活性真皮支架是实现促血管活性因子持续表达的有效手段。.主要研究内容：通过负载编码VEGF和促血管生成素-1(Ang-1)的PEI/多质粒DNA(pDNAs)纳米复合粒子，制备了双基因激活真皮支架(S2)。以同样的方式，制备了另一种装载pVEGF/Ang-1嵌合质粒的双基因活性真皮支架（S1）。在利用大鼠皮下埋植支架移植模型确定较优负载量之后，通过体外接种脐静脉内皮细胞及全层皮肤缺损模型移植实验，研究该基因活性支架的转染效能以及介导血管化的有效性，通过组织病理学检测、电子显微镜、分子生物学手段等方法，进一步揭示双基因活性支架的作用机制及信号通路可能的重要分子。.重要结果及关键数据：成功构建了2种双基因活性真皮支架，两种支架均能持续释放DNA复合物，并表现出有效的转染能力，能够在体外上调VEGF和Ang-1的表达。细胞实验表明，两种支架在体外均能促进成纤维细胞的增殖、迁移、人脐静脉内皮细胞的迁移和血管的形成。动物实验显示，与C2和S1相比，S2显示出显著更高的新生血管形成率、更好的血管成熟和伤口愈合。结果表明，与单基因激活的支架相比，双基因活性支架具有更大的促进血管生成的潜力。此外，不同加载方式的双基因活性支架在血管生成水平上也存在差异，加载两个基因的效果优于加载嵌合基因。.科学意义：双基因活性支架能够持续上调VEGF和Ang-1的表达，有望成为一种具有激活血管化功能的新型功能性组织工程皮肤。