HAEC-脱细胞结膜基质动态三维构建结膜植片及其体内转归机制

Conjunctiva is very important for maintaining ocular surface integrity and tear film stability. Conjunctival reconstruction is a prerequisite for successful ocular surface repair. At present, there is no ideal graft material instead of conjunctiva. Our previous study found that human amniotic epithelial cells (HAEC) could be induced to transdifferentiation into "conjunctival-like cells". preliminary exploration by phospholipase A2 and preparation of acellular conjunctival stent induced apoptosis method retains the collagen structure and mechanical properties of natural conjunctival stroma. Accordingly, we would use HAEC trans- differentiated conjunctival cells as seed cells, and acellular rabbit conjunctiva matrix as scaffold materials, and further cross-linked growth factor hierarchical release microspheres, which could restore the physiological microenvironment of acellular matrix scaffold to the greatest extent. The self-assembled nano peptide gel would be used to multilayers cells, and the physiological environment of conjunctival tissue would be simulated by three-dimensional dynamic culture system. Then, functional conjunctival graft would be quickly constructed in vitro. In addition, animal models of conjunctival defects would be established. Conjunctival grafts would be used to observe the survival and histocompatibility of the graft, and to explore the mechanism of exogenous HAEC, matrix remodeling and immunomodulatory mechanism. The result of this study would provide a safe, effective and transplantable conjunctival graft for clinical practice. (the key technologies involved in the project have declared 5 patents for inventions, 2 authorized)

结膜对维持眼表完整性和泪膜稳定性至关重要，结膜重建是成功进行眼表修复的先决条件，目前还缺乏代替结膜的理想移植材料。我们前期研究发现人羊膜上皮细胞（HAEC）可以体外诱导转分化为“类结膜细胞”；初步探索了利用磷脂酶A2和诱导凋亡法制备兔脱细胞结膜支架，保留了天然结膜基质胶原蛋白结构和力学性质。据此，本研究拟以HAEC转分化的“类结膜细胞”为种子细胞，脱细胞的兔结膜基质为支架材料，进一步交联生长因子分层缓释微球，最大程度的恢复脱细胞基质支架的生理微环境；联合自组装纳米多肽凝胶进行细胞复层，通过三维动态培养体系模拟结膜组织的生理环境，体外快速构建具有功能的结膜植片。建立结膜缺损动物模型，进行结膜植片移植，观察植片的存活情况及其组织相容性，并探讨外源性HAEC转归机制、基质重塑机制、免疫调节机制。本研究以期为临床提供安全、具有功效、可移植的结膜植片。（涉及的关键技术已申报发明专利5项，授权2项）

由于可用于移植的自体结膜极其有限，具有良好组织配型的异体结膜的获得相当困难，为了减少对正常组织的损害，解决结膜移植材料短缺及免疫排斥反应等难题，本项目探索利用人羊膜上皮细胞经体外诱导的“结膜样细胞”作为种子细胞；以异种脱细胞结膜基质作为构建载体，并在其上建立生长因子缓释系统；结合纳米多肽快速三维重建技术和动态气液培养体系，探索组织工程结膜重建的可行性。.经过本项目研究的开展，顺利建立了人羊膜上皮细胞向结膜上皮细胞转分化的共培养体系，进一步地，结膜上皮细胞在CHIR-99021的诱导下形成三维细胞球，且黏蛋白Muc5AC表达增强，解决了组织工程结膜重建的细胞来源问题；其次，利用磷脂酶A2精密脱细胞方法制备脱细胞结膜基质材料，其天然胶原成分及超微结构得到完好保存的，力学强度满足生理移植需求；在脱细胞结膜基质上以天冬氨酸（Asp）为交联臂，建立了EGF缓释支架DRC-Asp-EGF；利用纳米水凝胶的自组装特性，培养动态培养系统，在脱细胞结膜基质上种植种子细胞实现三维组织工程结膜的快速重建；在体移植实验结果表明，交联生长因子的脱细胞结膜基质可以促进结膜上皮修复，减少结膜瘢痕性修复，促进迁移细胞向结膜杯状细胞分化，重建损伤结膜的分泌功能。.本研究结果论证了DRC-Asp-EGF可能是一种有效的结膜重建替代品。进一步地，通过体外培养活性良好的种子细胞，可在脱细胞结膜基质上完成了复层组织工程结膜的快速体外重建，为结膜损伤治疗，修复损伤结膜功能提供了一个新的再生修复移植产品方案。