ADSCs通过MMP-1/PAR1信号通路调控KCs增殖和迁移的机制及其在构建仿生人工皮肤的应用

The main defect of the present tissue engineering skin including insufficient mechanical strength and poor migration and proliferation ability of KCs has limited its clinical application. The literature and our previous study found that adipose derived stem cells (ADSCs) could promote the migration and proliferation of KCs, but its mechanism still remained unclear. Besides, the nanofibrous scaffold with incorporating bioactive molecules or varying topological structure and phychemical properties could significantly influence the biological behavior of the cells, but there is no research on the regulation of KCs through directly regulating by nanofibrous scaffold or through ADSCs with scaffold. Based on this, the team proposed that the scaffold prepared by phychemical and biological modification is expected to directly regulating the proliferation and migration of KCs or through ADSCs, and further construct a novel tissue engineering skin which could be clinically applied. This project intends to further study the molecular mechanism in which ADSCs regulate the migration and proliferation of KCs, and accordingly incorporate bioactive molecules which could promote the function of KCs in combination with the topology structure and the phychemical and biological modification of nanofibrous scaffold to directly promote proliferation and migration of KCs or through ADSCs, and then fabricate novel tissue engineering skin via layer-by-layer technique and verify its effect on wound repair within skin defect model of nude mice. This project can further reveal the mechanism in which ADSCs could regulate the biological behavior of KCs, and successfully construct a novel tissue engineering skin which has important theoretical and clinical significance.

现有组织工程皮肤存在机械强度不足和角质形成细胞（KCs）迁移及增殖能力差等缺陷，限制了其临床应用。我们前期研究发现脂肪干细胞（ADSCs）可促进KCs的迁移和增殖，且ADSCs可以旁分泌大量MMP-1；而纳米仿生支架负载生物活性分子或者改变拓扑结构及理化特性可明显影响细胞的生物学行为。基于此，项目组拟深入研究ADSCs通过旁分泌MMP-1促进KCs增殖和迁移的机制，并筛选出发挥重要作用的关键分子；研究支架材料的拓扑结构和理化特性对ADSCs、KCs的细胞生物学行为的影响；根据ADSCs促进KCs迁移和增殖的研究结果，于支架材料内负载MMP-1或其下游发挥作用的关键活性分子，利用细胞层层叠加自组装的技术构建复层活细胞仿生人工皮肤，并在裸鼠皮肤缺损模型验证其修复功能。本项目可进一步揭示ADSCs对KCs的调控机制，并有望成功构建新型组织工程皮肤，具有重要理论及临床意义。

上皮化速度缓慢与机械强度不足成为限制组织工程皮肤应用的关键问题。项目组首先探究了ADSCs通过旁分泌MMP-1促进Kcs增殖和迁移的分子机制，并筛选出下游关键调控分子PKC；在阐明支架材料的拓扑结构、理化特性与负载PKC分子对ADSCs、Kcs的细胞生物学行为的影响的基础上，利用支架-细胞层层组装技术制备仿生人工皮肤，并利用小香猪皮肤缺损模型验证其在体功能。结果表明：（1）与对照组相比，MMP-1刺激组可显著促进Kcs增殖与迁移，加入其抑制剂FN439可逆转这一结果；MMP-1刺激可促进Kcs内PAR1蛋白表达，加入FN439可显著抑制PAR1表达，表明MMP-1对PAR1具有调控作用；（2）KCs中PAR1抑制对下游分子ERK、PKC表达水平产生显著影响，蛋白酶C(Protein kinase C，PKC)蛋白抑制剂处理组可显著降低细胞增殖与迁移，因此PKC可能为调控Kcs增殖与迁移的关键下游信号分子；（3）利用高分子络合法将PKC蛋白负载至PCL-CA复合纤维支架上，支架呈粗纤维-超细纤维交叉结构，孔径显著增大；（4）制备的新型PCL-CA-PKC纳米纤维膜可显著促进Kcs与ADSCs细胞的粘附与增殖，支架取向性可显著影响Kcs内部肌动蛋白分布，纤维-Kcs复合物可于7d内形成细胞膜片；（5）利用手持静电纺装置于5min内构建5层细胞/纳米纤维交替叠加的三维结构，于1周内初步构建出完整性良好的仿生活性皮肤；（6）仿生皮肤处理组可显著促进创面愈合与血管新生，仿生皮肤组的表皮化良好，接近正常皮肤；新生皮肤的胶原纤维排列更加整齐，表-真皮结构与基底膜完整且清晰。本课题研究成果不仅本课题研究成果不仅为构建实现缺损皮肤永久性替代的新型生物材料提供了理论基础与技术支撑，而且通过阐明MMP-1/PAR1信号通路对KCs增殖和迁移的作用机制，促进了再生医学及智能生物材料领域研究的发展。