诱导多能干细胞来源的视网膜色素上皮细胞和光感受器细胞移植治疗视网膜色素变性的实验研究

Retinitis pigmentosa (RP) is the most common inherited human eye disease resulting in night blindness and visual defects. This disease is caused by degeneration of the retinal pigment epithelium and apoptosis of the photoreceptors which lead to progressive visual loss. However, it remains incurable by now. Induced pluripotent stem cells (iPSCs), generated from somatic cells, are shown to be very similar to embryonic stem cells in morphology, proliferation and differentiation capacity. Thereby, iPSCs can meet the defeciency of transplanted cells without immunological or ethical problems. In this study, mouse iPSCs were induced via retroviral and electroporational induction of the transcription factors OCT3/4, SOX2, KLF4 and C-MYC in mouse skin fibroblasts (SFs). Then, iPSCs were induced to differentiate into retinal pigment epithelium and photoreceptors with definite no serum two-stage cultivation and definite no serum small molecule-based 3D cultivation respectively. After that, iPSCs-derived retinal pigment epithelium or/and photoreceptors were injected into the subretinal space of RP mouse models, and the effect was further evaluated by morphology, optokinetic testing, functional anatomy, electroretinogram and immunohistochemistry. We believe this study will provide experimental evidence and theoretical basis for clinical application of iPSCs-derived retinal pigment epithelium and photoreceptors in treating retinal degenerative diseases including RP.

视网膜色素变性（RP）是常见遗传变性性致盲眼病，视网膜色素上皮细胞（RPE）变性和光感受器细胞凋亡是其主要原因。目前对RP尚无有效治疗方法。诱导多能干细胞（iPSCs）具有增殖能力强、多向分化潜能高，避开了胚胎干细胞的伦理问题，克服了免疫排斥等重大医学难题，更解决了移植细胞来源的关键问题。本课题将OCT3/4、SOX2、KLF4、C-MYC四个重编程关键基因采用电穿孔和病毒转染小鼠皮肤成纤维细胞制备iPSCs并鉴定。拟采用特定无血清两阶段诱导培养法以及特定无血清小分子化合物3D培养法分别诱导iPSCs分化为RPE和光感受器细胞，并检测其功能。诱导分化的RPE和光感受器细胞经流式细胞仪分选纯化后，单种或两种细胞联合移植至RP鼠动物模型，探讨其治疗RP的可能性及疗效，为临床应用iPSCs诱导分化的REP和光感受器细胞移植治疗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）是中国乃至全世界不可逆性致盲眼病，而视网膜色素上皮细胞（REP）和光感受器细胞变性凋亡是其主要病因。本项目采用病毒转染的方法将四个重编程关键基因（OCT3/4、SOX2、KLF4 和C-MYC）导入小鼠尾端成纤维细胞。细胞经免疫细胞化学、Real Time PCR、多向分化和畸胎瘤实验，证实获取的细胞系具有干细胞特征，即诱导多能干细胞（iPSCs）。采用特定无血清两阶段诱导培养法将iPSCs向视网膜色素上皮细胞诱导分化后，扫描电镜显示细胞具有微绒毛结构，另外经定量PCR、免疫细胞化学和Western Blot等技术检测，诱导后的细胞表达视网膜色素上皮细胞特异性标记物。因此，无血清两阶段诱导培养法可以成功将iPSCs诱导分化为视网膜色素上皮细胞，且诱导分化效率较高。采用光感受器细胞共培养3D诱导培养法联合特定小分子化合物将iPSCs 向光感受器细胞诱导分化后，扫描电镜显示细胞具有外节结构，另外经Real Time PCR、免疫细胞化学和Western Blot等技术检测，诱导后的细胞表达光感受器细胞特异性标记物。因此，光感受器细胞共培养3D诱导培养法联合特定小分子化合物可以成功将iPSCs诱导分化为光感受器细胞，且诱导分化效率较高。将诱导分化后的视网膜色素上皮细胞和光感受器细胞通过视网膜下注射和玻璃体腔注射的方式，移植至RP 模型动物，通过形态学观察细胞移植情况和视觉电生理检测移植后视网膜功能。这为临床上应用iPSCs 诱导分化来源的视网膜色素上皮细胞和光感受器细胞移植治疗RP 等视网膜变性疾病提供实验基础和理论依据。此外，本项目还通过采用与角膜基质细胞或角膜内皮细胞共培养方式，将iPSCs 向角膜上皮细胞或角膜内皮细胞诱导分化后，发现诱导分化的细胞表达角膜上皮细胞以及角膜内皮的标记物。这为组织工程化角膜以及角膜眼表疾病的治疗提供了新思路。

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