LY2109761培养的角膜内皮细胞用于组织工程角膜内皮重建的研究

Corneal endothelial keratoplasty is the main treatment for corneal endothelial blindness. However, donor shortage has seriously limited the development of the surgery. Therefore, tissue engineered endothelium emerges as the times require, and the key of this method is to obtain ideal corneal endothelial cells (CECs). However, the spontaneous endothelial-mesenchymal transition (EnMT) during the ex vivo culture of the CECs always leads to the loss of the cell morphology and cell function, which is the bottleneck of this kind of research. Our preliminary study used LY2109761, a novel transforming growth factor-beta type I and type II receptor inhibitor, to culture the endothelial cells, and obtained LY2109761-CECs that maintained the phenotype in vitro. At the same time, we successfully seeded CECs on decellularized corneal stroma using “silicone tube cell seeding model” designed by ourselves. On this basis, the research group will further compare the differences of each passage of CECs cultured with different concentrations of LY2109761 regarding the expression of functional markers, mesenchymal markers and proliferative ability, in order to find the optimal seeding cells. Additionally, we will construct an endothelial graft with chosen seeding cells and decellularized thin stroma and test its mechanical and biological properties in vitro. We will also use rabbit as animal model to examine the structure and function of the graft. We expect to achieve an endothelial graft, which is biocompatible and functional in vivo. We hope to find a new organ source for patients who need endothelial keratoplasty, and strive for clinical translation.

内皮移植术是治疗角膜内皮盲的主要手段，但供体短缺严重制约其开展。组织工程内皮重建应运而生，其关键是获得大量理想的角膜内皮细胞（CECs），而CECs体外培养出现间质化，致其结构功能丧失是目前亟待解决的研究瓶颈。课题组前期使用TGF-β受体I/II双重抑制剂LY2109761体外培养内皮细胞，成功获得保持内皮形态的CECs；同时使用自主研发的“硅胶管模型”完成了CECs在无细胞角膜基质上的种植。在此基础上，本课题组将进一步针对抑制内皮间质化这一难题进行攻关，完善对不同浓度LY2109761处理的CECs的鉴定，比较各代细胞功能蛋白、间质蛋白表达和增殖活性的差异，选取形态良好、功能和增殖活性蛋白表达均较高的CECs为种子细胞，进而利用其和无细胞薄层基质构建内皮植片，在体外检测植片的性能。随后以兔模型研究植片在体内的结构和功能，期望获得一种生物相容性好、体内功能明确的内皮植片，并争取向临床转化。

角膜内皮移植术是治疗内皮盲的主要手段，但供体短缺严重制约其开展。组织工程内皮重建应运为生，其关键是获得大量理想的角膜内皮细胞(CECs)，而CECs在体外培养易出现间质化，致其结构功能丧失是目前亟待解决的研究瓶颈。课题组研究发现利用TGF-beta受体I/II双重抑制剂LY2109761在体外培养CECs，可保持内皮细胞的形态和功能。课题组在CECs传代时，分别用含有不同浓度LY2109761(0.1、1和10μM)的培养基培养CECs，并与未加入LY2109761培养的CECs相比较，观察细胞形态，并完成细胞增殖实验（MTT）。结果显示LY2109761培养的角膜内皮细胞(LY2109761-CECs)可在体外稳定传代，其细胞形态明显优于直接分离培养的CECs。MTT实验中，10uM组、1uM组、0.1u M组及对照组细胞在48h后OD490nm的吸光值不存在统计学差异，而在72小时后10uM组与1uM组、0.1uM组及对照组细胞在OD490nm的吸光值存在统计学差异。因此，实验组选择LY2109761浓度为1uM进行后续实验。接下来实验组利用1uM的LY2109761处理的CECs和对照组CECs并利用免疫组化和PCR的技术进行鉴定，主要比较细胞功能性蛋白包括离子泵蛋白(Na+/K+-ATPase)和闭锁小带蛋白1(zona occludens-1，ZO-1)的表达。同时检测间质类蛋白CD73、vimentin和fibronectin在两组细胞中的表达情况。结果显示：功能性蛋白包括离子泵蛋白(Na+/K+-ATPase)和闭锁小带蛋白1(zona occludens-1，ZO-1)的表达在LY2109761-CECs中明显升高，而间质类蛋白vimentin和fibronectin的表达明显减低，说明LY2109761能维持CECs的正常形态和功能，并能有效抑制CECs细胞在体外连续培养时的纤维化。利用LY2109761培养CECs，可在体外连续培养获得大量形态功能极佳的内皮细胞。利用LY2109761-CECs做为种子细胞进行组织工程角膜内皮重建，能大大缓解供体短缺的压力，为更多需要角膜移植的患者带来新的希望。研究成果已发表于《Cell Physiol Biochem》2018。