基于嵌合核酸适配体构建细胞坏死响应性材料实现主动调控血管新生

One of the major challenges of tissue engineering is how to ensure the integrative fusion of the transplanted grafts (cell and biomaterial) with the host vascular network and the establishment of the neo blood supply in order to promote tissue regeneration and ultimately replace the functions of the necrotic tissues. The regulation of on-demand angiogenesis in the necrotic tissue is cutting-edge issues for investigation. This proposal is the first attempt to take necrotic cells / tissues secreted ATP, as a response signal, which are instantly translated into biological signals of angiogenic growth factor (PDGF), thus achieving the active and directional induction of angiogenesis and establishment of the vascular network. Chimeric ATP-PDGF aptamers are innovatively designed to realize the ATP-responsive release of PDGF growth factors from a hydrogel-based biomaterial, which recapitulates responsive release of angiogenic growth factor in vivo. Meanwhile, highly systematic and integrated plans are laid out to verify the effectiveness of this responsive smart material from molecular, material, cellular and in vivo levels respectively. In particular, micro-fabrication technology is adopted to create novel bilayered in vitro model for co-culture to imitate the multilayer structure of the native vasculature, which realize the biomimetic in vitro angiogenesis with feedback on cell necrosis. The instructive angiogenesis based on this responsive smart material is expected to promote repair and regeneration of necrotic cells/ tissues, and further assist the integration and survival of the transplanted tissue engineering products with the host vascular network.

组织工程重大挑战之一是如何保证移植物（细胞/材料）与体内血管网融合，建立新生血供从而促进组织再生，最终替换坏死组织功能。调控血管新生，使其在需要血供再生的坏死组织中可控实现是亟待解决的前沿问题。本计划首次尝试将坏死细胞/组织分泌的ATP作为响应信号，即时即地转化为促血管新生生长因子（PDGF）的生物信号，实现主动式、指向性地诱导血管新生。通过创新性的ATP-PDGF嵌合适配体设计，实现ATP响应下PDGF从水凝胶材料中的可控释放,模拟体内响应性释放生长因子的促血管再生。同时提出高度系统和整合的方案从分子、材料、细胞和活体各水平上验证此智能材料的功效。其中在细胞水平上结合微加工技术建立新型仿血管多层结构的双环共培养体外模型，实现对细胞坏死反馈的体外血管新生仿生模拟。基于此响应智能材料实现的指向性血管新生预期会促进坏死细胞/组织的修复与再生，并辅助组织工程产品移植后与宿主血管网的融合和

本研究计划中设计了新型适配体修饰的生物材料，尝试开发对于体内坏死微环境中ATP响应的核酸适配体修饰生物材料，实现血管新生，实现促进坏死细胞、组织的修复与再生，并进一步帮助移植后组织工程产品在体内与宿主血管网的融合和存活。项目进行的四年中，依照项目计划的各个方向进行，分别取得了较好的成果。此外，根据领域内最新进展和最新需求将项目目标进一步扩增为不仅开发出促血管再生材料，并且利用促血管再生材料可辅助干细胞治疗下肢缺血性疾病和皮肤修复，同时对此高性能3D材料发挥作用的机制进行了探索。主要取得了以下四方面的阶段性成果：一、开发可以对细胞组织坏死释放的ATP响应的适配体设计技术，并通过滚环扩增（RCA）技术实现响应适配体扩增、修饰，并且基于适配体修饰基地材料实现了ATP的无标记检测。二、对上述RCA适配体扩增、修饰技术进行了应用，开发多价核酸适配体用于靶向细胞的富集，为个体化医疗靶向细胞富集提供一种新的技术手段。三、促血管再生材料辅助干细胞治疗实现高效下肢缺血性疾病和皮肤修复的治疗。四、解释了三维促血管再生材料优于二维培养系统的机制。本项目的实施对于设计新型基于核酸适配体的智能响应性生物材料，及其在医疗检测和再生医学领域的应用具有较高的理论和实际应用价值。项目产生的相关成果发表了11篇高水平论文（申请人为通讯作者），其中RCA技术扩增适配体平台技术和材料修饰相关领域发表3篇文章(Biosensor and Bioelectronics； Scientific Report; RSC Adv. )；促血管再生材料发表于Acta Biomaterials和美国科学院院刊（PNAS）上，相关工作引起国内外广泛报道；皮肤修复相关文章发表在Advanced Healthcare Materials上; 机制探索相关工作在知名期刊Biomaterials发表。同时在新型适配体修饰检测平台和促血管新生新型生物材料两个创新技术上分别获得三项中国专利授权。

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