基因活性真皮支架调控血管生成素表达及介导早期血管化的研究

Slow angiogenesis of dermal substitutes at the early stage is still a severe problem that should be solved urgently. Angiogenic factors play an important regulation role in the process of angiogenesis, which has also been proved by our previous studies. How to realize the effective delivery and sustained impact of angiogenic factors is the key. The emergence of gene-activated scaffolds (GASs) provides a new model to effectively deliver and controllably release angiogenic factors. The genes loaded in the GASs can be naked DNA, also be gene vector/DNA complexes. Angiogenin (ANG), with the strong ability to promote angiogenesis, can be used to construct the gene-delivery system of GASs. In this system, sustained transfection occurs in the local wound site, and ANG can be encoded and released to enhance the process of angiogenesis, indicating a promising method to substantially solve the problem of insufficient angiogenesis. Based on the previous study results, this study will be proposed to prepare a novel ANG gene-activated dermal scaffold by incorporating the nano-scaled complexes of cationic gene vectors and ANG plasmid DNA into PLGA knitted mesh-reinforced collagen-chitosan porous scaffold (PLGAm/CCS). PLGAm/CCS has been proved to promote angiogenesis and induce in situ tissue regeneration in the one-step repair of full-thickness skin defects, indicating its potential in the skin tissue engineering. The roles of the ANG gene-activated dermal scaffold in the early-stage angiogenesis, as well as the relevant angiogenesis mechanisms, will be investigated The results will provide a new solution for promoting angiogenesis, and also provide more theoretical bases for the development of novel dermal substitutes.

真皮替代物早期血管化不足的问题亟待解决。血管生成素（Angiogenin，ANG）参与血管从新生到成熟的整个过程，并发挥基础性的正性调控作用，前期研究证实ANG早期的促血管化作用，但如何实现有效传递和局部持续作用是关键。基因活性支架（GASs）的出现使促血管活性因子的传递和释放出现更高效的作用模式。将GASs作为基因传递体系可以有效实现基因在创面局部持续释放，转染修复细胞并表达特定的促血管活性因子，解决真皮支架早期血管化不足的问题。本项目在前期基础之上，拟将基因载体包被ANG质粒DNA形成的纳米级复合微粒整合到PLGA编织网/胶原-壳聚糖真皮支架上获得新型GASs，通过体外灌流法接种脐静脉内皮细胞及全层皮肤烫伤模型移植实验，研究该活性支架的转染效能以及介导早期血管化的有效性，揭示ANG作用机制及信号通路，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理论依据。

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。血管生成素（ANG）参与血管从新生到成熟的整个过程并发挥基础性的正性调控作用，前期研究证实ANG早期的促血管化作用，但如何实现有效传递和局部持续作用是关键。基因活性支架（GASs）的出现使促血管活性因子的传递和释放出现更高效的作用模式。将GASs作为基因传递体系可以有效实现基因在创面局部持续释放，转染修复细胞并表达特定的促血管活性因子，解决真皮支架早期血管化不足的问题。.主要研究内容：本项目将基因载体壳聚糖季铵盐（TMC）包被ANG质粒DNA形成纳米级复合微粒，并将其整合到前期PLGA编织网/胶原-壳聚糖真皮支架（PLGAm/CCS）上获得一种新型GASs，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烫伤模型移植实验，研究该基因活性支架的转染效能以及介导早期血管化的有效性，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ANG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：成功构建了一种ANG基因活性真皮支架TMC/pANG-PLGAm/CCS，该支架具有一定缓释能力和较理想的转染性能，转染后的HUVEC细胞能够保持良好的增殖活性并分泌Angiogenin蛋白。基因负载量为5~10 μg的单位支架能有效促进支架细胞浸润、组织生长与血管新生和成熟。TMC/pANG-PLGAm/CCS可促进全层缺损创面愈合，将真皮支架血管化时间提前至移植术后第5天；其机制可能与Erk1/2通路、辅肌动蛋白系统有关。.科学意义：本研究结果为真皮支架早期血管化问题提供一种新的、基于基因治疗与强化型真皮支架的解决方案，为新型真皮替代物的开发提供理论依据。