基因修复的自体诱导多能干细胞来源的视网膜色素上皮细胞移植治疗视网膜色素变性的实验研究

Retinitis pigmentosa (RP) is a hereditary irreversible blindness disease. The structure and function of the pigment epithelium abnormalities resulting in photoreceptor cell apoptosis and necrosis is the major cause of this disease. Gene therapy, neuroprotective factor supplemental treatment and stem / progenitor cell transplantation all have some limitations, which seriously restrict their applications. Induced pluripotent stem cells (iPSCs) have more advantages, such as strong proliferative ability, multiple differentiation potential, no ethical problems, and no immune rejection when transplanted to host. It is an ideal cell source for the treatment of RP disease by cell transplantation. However, there are several problems need to be explored further. Consequently, in this study, RCS rat iPSCs will induced via electroporational induction of the transcriptional factors such as Oct4, Sox2 and Klf4 in urine-derived cells (UCs). By using CRISPR/Cas9 mediated site-specific genome engineering technology, the DNA sequence of the Mertk mutation site will modified and the mutation repaired iPSCs will gotten. The mutation corrected iPSCs will induced into retinal pigment epithelium cells by adding nicotinamide and either activin A or IDE-1 small molecule-based cultivation. The silk fibroin scaffolds will prepared and then iPSCs-derived RPE cells will be seeded onto the membrane of silk fibroin scaffolds to prepare the RPE cell membrane. Sheets of iPSCs-derived RPE cell membrane will be transplanted into subretinal space of RCS rat, which is the model of RP, by external-route microsurgery. The feasibility and efficacy of these methods for the treatment of RP will be explored and studied seriously. And the autologous iPSCs-derived RPE cells by gene repair transplantation for RP and other retinal degenerative diseases will then be clarified, which will provide a solid foundation both in experimental and theoretical basis for clinical application in near future.

视网膜色素变性（RP）是遗传性不可逆致盲眼病，视网膜色素上皮（RPE）结构与功能异常致光感受器细胞凋亡是其主要病因。基因治疗、神经保护及干/祖细胞移植均存在弊端，严重制约其应用。诱导多能干细胞（iPSCs）增殖能力强、多向分化潜能、无伦理问题，自体移植无免疫排斥等优势，是细胞移植治疗RP的理想细胞来源。然而，仍存在众多亟待解决的问题。本课题将三个重编程关键基因经电穿孔导入大鼠自体尿脱落肾小管上皮细胞制备iPSCs；采用CRISPR/Cas9技术校正突变Mertk位点DNA序列，获得基因修复的iPSCs；经无血清添加新的小分子化合物方法将修复的iPSCs分化为RPE细胞；制备蚕丝蛋白生物膜，RPE细胞种植其上制备细胞膜片；外路法自体RPE细胞膜片移植至RCS大鼠，探讨该方法治疗RP可行性和疗效，为临床应用基因修复的自体iPSCs来源RPE细胞移植治疗RP等视网膜变性疾病提供实验基础和理论依据

视网膜色素变性（RP）是全世界最常见的遗传性视网膜疾病，进行性加重不可逆致盲眼病，其特征是光感受器细胞变性和视网膜色素上皮细胞（RPE）损伤。本研究利用电穿孔方法将重编程关键基因，导入小鼠自体尿液脱落肾小管上皮细胞，成功制备自体iPSCs并鉴定。采用自发分化法将iPSCs诱导分化为视网膜色素上皮细胞并鉴定。制备蚕丝蛋白生物工程支架，并将诱导分化后的iPSCs-RPE细胞成功培养其上，制备可移植的RPE细胞膜片。碘酸钠方法制备视网膜退行性疾病C57BL/6小鼠模型，并分别采用细胞悬液法和细胞膜片法将RPE细胞成功移植入模型小鼠视网膜下腔，免疫组化、避光实验等功能检测和移植后的临床观测。为临床应用自体来源iPSCs 诱导分化的RPE移植治疗RP等视网膜退行性疾病，提供实验基础和依据，并具有巨大潜在临床应用价值。

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