可降解干细胞组织工程化视网膜膜片修复视神经病变的机制及疗效研究

The treatment of neural blindness (Glaucoma, AMD and so on) is a big worldwide challenge, and most of neural blindness patients have been diagnosed as the advanced/end stage in China. Stem cell replacement therapy has intrinsic problems, such as lower survival rate, inaccurate target, and difficult integration, no mention to visual function restore. This investigation focuses on stem cell and tissue engineering to explore the critical gene or signal pathway to provide higher proliferation and lower tumorigenesis seed cells，and then induce seed cells into retinal neurons . New degradable material PLGA is selected to make up tissue engineering retina scaffold and co-culture with stem cell-induced retinal neurons. The patch clamp has been adopted to precisely measure single cell / whole retinal patch eletrophysiologic potentials. Finally, the retinal patch is transplanted into the eyes of glaucoma and AMD-like monkey mode. The morphology and function evaluation are needed to offer a new insight into the mechanisms of target location，imigration & integration and function restore, and a new therapy to the patients with eye diseases of neural blindness.

神经性盲（如青光眼、老年黄斑变性等）的治疗是人类共同面临的尚待攻克的临床难题，我国大多数该类患者就诊时就已进入神经性盲的晚期，干细胞替代疗法不能有效治疗该类晚期病变患者，盖因存在宿主功能细胞少，移植细胞存活率低，难以靶向定位，且与宿主视网膜不易整合等不易克服的缺点。据此，本研究结合干细胞和组织工程技术，优选适宜的种子细胞，寻找诱导种子细胞分化为视网膜神经元的关键基因及信号通路，结合新型可降解高分子材料(PLGA)飞秒激光蚀刻技术构建“可降解干细胞组织工程化视网膜膜片”，用全细胞膜片钳技术检测单细胞层面及三维视网膜膜片整体层面的各朝向的电生理功能，靶向移植入恒河猴青光眼和类AMD动物模型眼内，探索其修复视神经损伤的机制及疗效，以期达到靶向移植且移植细胞存活率高，存活周期长、与宿主视网膜整合良好的目的，为临床治疗神经性盲患者提供崭新的治疗手段，改善、提高甚至部分恢复神经性盲患者的视功能。

本项目已完成预期计划，成功构建“可降解干细胞组织工程化视网膜膜片”。获得主要研究结果如下：1.通过控制分化过程中拟胚体直径和调控Wnt信号通路，干细胞种子三维视杯产效率提高20倍；改良程序降温冻存方案，成功实现类器官冷冻保存。2.三维视杯在PLGA支架上形成视网膜节细胞生长的轴突，并测得电生理活动。3. 应用临床级别地塞米松激素缓释药物，调控移植期眼球免疫系统。4. 视网膜膜片移植到灵长类恒河猴眼球，已达到靶向移植且移植细胞存活率高，存活周期长、与宿主视网膜整合良好的效果。结果初步证明视网膜膜片移植方法对神经性盲治疗是可行的，为完成临床前非人灵长类动物研究打下坚实的基础，研究达到国际先进水平。