黄芪多糖-胶原-丝素蛋白3D打印支架载VEGF165基因修饰iPS-HFSCs的移植在全层皮肤缺损修复中的效用及作用机制研究

Large area defect of the skin is one of the clinically refractory diseases. How to promote the early vascular regeneration of skin wounds remains unresolved.Previous studies in this project have demonstrated that astragalus polysaccharides-collagen-silk fibroin composite scaffolds can accelerate skin wound repair by promoting vascularization; iPSCs can be induced into HFSCs.However, the effect and internal mechanism of tissue engineered skin composed of VEGF165 gene-modified iPS-HFSCs combined with astragalus polysaccharides-collagen-silk fibroin 3D printed scaffold in the repair of full-thickness skin defects remains to be elucidated.Astragalus polysaccharide.Based on this, the project aims to focus on the nutritional blood supply in wound repair, using 3D printing, bioengineering scaffolding, iPS reprogramming, lentiviral packaging, real-time PCR, western blot and other technologies to further study the above-mentioned tissue engineering skin. The role of full-thickness skin defect repair and related molecular mechanisms.Providing ideas for the early vascularization of tissue-engineered skin,providing scientific experimental basis and new strategy for the treatment of skin defects with the new technology of combined TCM and western medicine, which has important research significance for further revealing the mechanism of wound repair and the development of regenerative medicine.

皮肤大面积缺损是临床难治性疾病之一，如何促进皮肤创面早期血管再生难题仍未解决。本项目前期研究已证明黄芪多糖-胶原-丝素蛋白复合支架可通过促血管化而加速皮肤创面修复；诱导多能干细胞（iPSCs）可诱导为毛囊干细胞（HFSCs）。然而，目前对VEGF165基因修饰的iPS-HFSCs复合黄芪多糖-胶原-丝素蛋白3D打印支架构成的组织工程皮肤在全层皮肤缺损修复中的效用及机制有待阐明。基于此，本项目拟聚焦创面修复中营养血供这一重点，采用3D打印、生物工程支架、iPS重编程、慢病毒包装、real-time PCR、western blot等技术，深入研究上述组织工程皮肤在全层皮肤缺损修复中的作用及相关分子机制。为组织工程皮肤的早期血管化提供思路，为中西医结合新技术治疗皮肤缺损疾病提供科学的实验依据与新策略，这对于进一步揭示创面修复的作用机制和再生医学发展具有重要研究意义。

背景: 制备可降解支架，负载细胞和因子是组织工程皮肤的特点。iPS细胞理论上具有无限增殖能力并在可分化成近乎所有成体细胞类型。因此，基于iPS细胞的组织工程皮肤治疗创面修复值得探讨。.方法: 收集健康人皮肤成纤维细胞，并重编程为iPS细胞。经过基因修饰和诱导，得到CK19+ /Integrinβ1+ /CD200+ VEGF165基因修饰的iPS-HFSCsGFP。采用免疫荧光、RT-qPCR法联合鉴定诱导的毛囊干细胞。制备含黄芪多糖的3d打印可降解支架，并将其与VEGF165基因修饰的iPS-HFSCsGFP共培养，通过CCK8检测和扫描电镜分析组织工程皮肤的生物相容性和空间结构。最后，将组织工程皮肤移植到裸鼠背部创面上，通过组织学、免疫组化、免疫荧光，RT-qPCR和双光子显微镜下活体三维重建法，联合评价组织工程皮肤对全层皮肤缺损再生修复的效果。.结果: 获得与人来源毛囊干细胞形态和表型接近的CK19+ /Integrinβ1+ /CD200+ VEGF165基因修饰的iPS-HFSCsGFP，。制备的含200μg/ml黄芪多糖的3d打印可降解支架表面富集蜂窝状网状结构，更有利于所得细胞的增殖。组织工程皮肤移植后，联合检测显示其促进了创面的早期血管化、胶原和毛囊再生，加速了创面修复。.结论: VEGF165基因修饰的iPS-HFSCsGFP复合含200μg/ml黄芪多糖的3D打印可降解支架可直接和间接参与皮肤中血管、胶原和毛囊的新生，实现较为完整的结构性和功能性皮肤再生修复。