转录因子诱导人脐带间充质干细胞转分化为视网膜色素上皮样细胞的研究

Transdifferentiation of human umbilical cord mesenchymal stem cells into retinal pigment epithelial like cells

目的探讨采用关键转录因子将人脐带间充质干细胞(human umbilical cord mesenchymal stem cells,h UC-M SCs)转分化为视网膜色素上皮(retinal pigment epithelium,RPE)样细胞的方法。方法通过流式细胞技术鉴定h UC-MSCs的表面标记分子;通过诱导h UC-MSCs分化成为脂肪、骨和软骨细胞确定其多系分化能力;采用慢病毒包装系统获得分别含有11个转录因子Sox2、Pax6、Rax、Six6、Nr2e1、Otx2、Lhx2、Crx、Mitf-A、Klf4和c-Myc的病毒,并通过感染h UC-MSCs来诱导h UC-MSCs向RPE样细胞分化。结果 h UC-MSCs表达CD29、CD44、CD73、CD90和CD105,但不表达CD11b、CD34,CD45和HLA-DR等分子标记,在成脂、成骨、成软骨分化培养基中可分化为脂肪、骨和软骨细胞。视网膜及RPE发育相关的关键转录因子能够将h UC-MSCs直接转分化为RPE样的细胞,并表达ZO-1、RPE65、Bestrophin-1(Best-1)、CK8/18、Cralbp、Mertk、Tyrosinase(Tyr)、PEDF等RPE细胞的特征分子。结论视网膜及RPE发育相关的关键转录因子可直接将h UC-MSCs分化为RPE样细胞。

Objective To investigate the transdifferentiation of human umbilical cord mesenchymal stem cells（ h UC-M SCc） into retinal pigment epithelial（ RPE） cells. Methods M SC specific markers were identified by flowcytometry in h UC-M SCs,and adipogenesis,osteogenesis and chondrogenesis were confirmed by culturing h UC-M SCsin differentiation media. Sox2,Pax6,Rax,Six6,Nr2e1,Otx2,Lhx2,Crx,M itf-A,Klf4 and c-M yc were co-infected into h UC-M SCs by lentivirus-mediated system. The h UC-M SCs derived RPE-like cells were characterized by detecting the expressions of specific markers by real-time quantitative-polymerase chain reaction（ Q-PCR）,immunostaining and Western blot. Results h UC-M SCs were positive for CD29,CD44,CD73,CD90, CD105 and negative for CD11 b,CD34,CD45,HLA-DR. After induction,h UC-M SCs were differentiate into adipocytes,osteoblasts and chondrocytes. Retina and RPE development related transcription factors directly transdifferentiated h UC-M SCs into RPE-like cells which expressed RPE specific markers ZO-1,RPE65,Bestrophin-1,CK8 /18,Cralbp,M ertk,Tyrosinase and PEDF. Conclusion h UC-M SCs could be directly transdifferentiated into RPE-like cells by key transcription factors.