仿生平台生物打印及构建角膜抵抗细胞成肌纤维化变异研究

Three-dimensional (3D) bioprinting based corneal tissue engineering (TE) and the transplantation is a new measure for the treatment of refractory corneal diseases. However, how to resist the cellular myofibroblast deformation for the bioprinting construct is still a difficult problem. In this project, corneal stromal layer is constructed by compression and traction of collagen microenvironment as well as human or rabbit corneal stromal cells (CSCs) laden and orthogonal-multilayer stack. The corneal epithelial layer and endothelial layer are constructed by spheroid bioprinting human or rabbit corneal epithelial cells (CEpCs) and corneal endothelial cells (CEnCs) onto collagen membrane or thin accellular corneal matrix. The whole cornea is constructed by the biomimetic platforms, in which corneal stroma construction is induced by compression and traction collagen microenvironment, spheroid CEpCs and CEnCs are bioprinted on the surface and base of corneal stromal TE and such bioprinting corneal constructs are co-cultured in perfusion bioreactor. The bioprinting constructs of rabbit layered and whole corneas by the biomimetic platforms are used as donors to transplant and repair the corneal stem cells (LSCs), CEnCs and CSCs dysfunction rabbit models. The tests of cell morphology, phenotype, gene differences, high-throughput bioinformatics analysis of RNA-Seq transcriptomics, multiple signaling pathways, electric potential differences of endothelial cells, histology and so on are carried out. The molecular biological mechanisms of biomimetic platforms bioprinting and constructing corneas for the resistance of cellular myofibroblast deformation in three kinds of corneal cells are explored. This project can uncover the effect on the corneal repair and regeneration as well as correlation after corneal transplantation using bioprinting corneal constructs. And the project can also provide new ways and theoretical basis for the therapy of corneal diseases.

3D生物打印构建角膜及其移植是治疗难治性角膜疾病新的手段，但打印后构建体的细胞如何抵抗成肌纤维化变异仍是难题。本项目通过压缩和牵拉胶原微环境，正交多层叠加构建含角膜基质细胞(CSCs)有序排列基质层。利用角膜上皮细胞(CEpCs)和角膜内皮细胞(CEnCs)球形打印在胶原膜或脱细胞角膜薄片上构建上皮层及内皮层。应用压缩和牵拉胶原引导基质构建、表面与底面CEpCs与CEnCs球形打印、灌注生物反应器共培养仿生平台构建全层角膜。以此构建的分层角膜和全层角膜为供体，行角膜移植修复角膜三种细胞功能障碍兔动物模型。进行细胞形态与表型、基因差异、RNA-Seq转录组学高通量生物信息学分析、多信号通路、内皮电位差、组织学等检测，阐明仿生平台生物打印及构建角膜抵抗角膜三种细胞的成肌纤维化变异的分子生物学原理。揭示其角膜移植后对角膜细胞损伤修复以及对角膜再生的作用。为治疗角膜病变提供新的手段和理论依据。

1. 项目背景：.角膜病是主要的致盲性眼病，3D生物打印构建角膜及其移植是治疗难治性角膜疾病新的手段，但打印后组织工程角膜的细胞如何抵抗成纤维化变异仍是难题。.2. 主要研究内容：.通过压缩、牵拉胶原微环境及正切多层有序叠加排列构建，获得抗纤维化(KFM)的CSCs组织层。球形生物打印构建CEpCs细胞片与CEnCs组织片，获得抗纤维化(EMT)角膜上皮细胞层与内皮细胞层。结合机械拉伸及球形生物打印仿生打印构建板层组织工程角膜，进行生物反应器培养，并进一步开展体内外研究。.3. 重要结果、关键数据：.(1)球形培养、生物反应器系统有效促进Muse干细胞分化为CSCs；iPS-CM中的Actvin A等可上调CEnCs的PI 3-kinase信号通路。这些手段具有抗CSCs的KFM和CEnCs的EMT作用。(2) LM22B-10小分子可以促进角膜神经再生，其作用与CSCs-CEpCs对话、ERK-CREB通路激活有关。(3)角膜基质提取物和低浓度胎牛血清、EZH2抑制、高钾有利于抗KFM的人CSCs培养，获得大量生理型CSCs细胞，有利于细胞移植治疗角膜纤维化。(4)结合Y-27632、双培养基和球形培养的联合应用可获得高质量生物墨水，在精确度可达0.1 mm的自组装生物球形打印机精准地将CEpCs细胞球按图案自动打印到胶原膜，获得仿生CEpCs层。(4) 双向拉伸胶原可获得高机械和光学性能的仿生CSCs层，结合生物打印和灌流培养构建出仿生角膜前板层(含抗EMT的CEpCs和抗KFM的CSCs)，移植后治疗兔角膜损伤动物模型，可以显著促进角膜损伤修复，具有抵抗角膜纤维化的效果。.4. 科学意义：.阐明仿生平台生物打印及构建角膜抵抗角膜三种细胞的成纤维化变异的分子生物学原理。通过仿生平台构建组织工程角膜，移植后改善角膜功能，为解决3D生物打印构建角膜难题提供新的手段和理论依据。具有重要的临床和社会意义。