自体iPS来源的RPE移植后眼炎症反应的机理和干预研究

Autologous transplantation of induced pluripotent stem cells (iPS) derived retinal pigment epithelia (RPE) is currently the most promising therapy for RPE-related retinal diseases. However, maintaining the long term survival of transplanted cells remains the major challenge for successful RPE transplantation. Cell death and ocular inflammation are commonly associated with RPE cell therapy. However, the mechanisms of the death of iPS-RPE and the related ocular inflammation remain largely unknown. We found previously that the long-time suspended iPS-RPE failed to attach to the basement membrane and resulted in pyroptosis that is associated with increased mitochondrial fission and down-regulation of Integrin-FAK signaling. Moreover, we found that pyroptic cells released numerous damage-associated molecular patterns (DAMPs) that consequently exacerbated ocular inflammation. We hypothesize that the initial death of a fraction of transplanted cells triggers ocular inflammation, which in turn results in inflammatory attack to the transplanted cells and subsequent death of the transplanted cells. Therefore, stopping the vicious circle of inflammation and cell death would be an essential strategy for the long-term survival of transplanted cells. To test this hypothesis, we aim to identify the mechanisms of transplant-induced iPS-RPE pyroptosis and its relationship to ocular inflammation. The specific aims include: (i) exploring the role of mitochondrial fission in iPS-RPE pyroptosis; (ii) evaluating the detrimental effects of DAMPs as well as the protective effects of integrin/FAK signaling activation on pyroptic iPS-RPE; and (iii) assessing the suppressive effects of autologous serum derived laminin vector and gene therapy on iPS-RPE pyroptosis. Successful completion of this project would provide new insight into the mechanisms of ocular inflammation induced by iPS-RPE transplantation and inflammatory cell death. In addition, the outcome of this study may lead to a new therapeutic strategy using iPS-RPE transplantation to treat retinal diseases.

自体诱导多能干细胞来源的视网膜色素上皮细胞（iPS-RPE）是治疗视网膜病变的新希望，然而其移植后大多死亡并导致炎症。虽然我们前期研究发现了iPS-RPE移植后长期悬浮于视网膜下腔，整合素-局部黏着斑激酶（FAK）信号通路下调，线粒体裂解增加并发生程序化炎性坏死，促进损伤相关模式分子（DAMPs）释放，加剧炎症反应及组织损伤，然而iPS-RPE细胞程序化炎性坏死的具体调控机理及对其他细胞的影响尚不清楚。由此，本课题拟开展如下研究：（1）iPS-RPE细胞线粒体裂解诱导的程序化炎性坏死的调控机理；（2）iPS-RPE细胞坏死释放的DAMPs对其他细胞的损伤及整合素-FAK信号的保护机制；（3）自体血清层粘蛋白膜载体及基因靶向调控抑制iPS-RPE细胞程序化炎性坏死的干预研究。本课题可望阐明自体iPS-RPE移植后眼炎症反应的机理，为细胞移植治疗眼病提供新思路和新手段。

视网膜退行性疾病具有不可逆性致盲，以RPE死亡为主要特征，目前尚无有效治疗方法。自体iPS来源的RPE细胞移植替代治疗为该类疾病带来新希望。然而，RPE细胞体内移植后，如何减少炎性坏死，提高存活率，并长期发挥作用？目前仍是一项挑战性课题。本项目首先获得大量iPS-RPE细胞，解决细胞来源问题，借助iPS分化3D视网膜体系，我们优化出两条iPS-RPE来源途径：从2D分离培养原代RPE和3D视网膜富集RPE球，可以获得大量高纯度的iPS-RPE并具有功能性，同时建立了单细胞测序平台，系统地分析了iPS分化来源视网膜细胞转录组图谱。其次，我们还探讨了RPE在氧化应激环境中的各种死亡模式，以及阐明NLRP12/NLRP3/NLRP6在眼内炎症中的作用机理。最后，通过构建新型生物膜辅助提高iPS-RPE体内移植存活率，新型生物膜具有良好细胞相容性，免疫原性低，受体组织结构相符以及可以维持RPE极性等。本项目获得的iPS-RPE制备方法和iPS-RPE细胞生物膜片，有望进行GMP级别制备并应用于临床。