人视网膜前体细胞的体外视网膜组织片下移植

Transplantation of human retinal progenitors under cultured neural retina in vitro

目的研究人视网膜前体细胞移植到体外培养的人视网膜组织片下的细胞分化。方法取无眼部发育异常的4～5个月胚胎眼球，进行视网膜前体细胞分离培养。将传代的细胞移植到体外培养的视网膜神经上皮组织片下，通过光学显微镜和免疫组织化学观察细胞分化和组织整合情况。结果人视网膜前体细胞在体外培养时形成神经球样细胞团，传代后形成子代细胞团，表达神经干细胞标志Nestin。体外培养的视网膜组织片在5d、10d均能基本维持视网膜结构。移植到视网膜组织片下的视网膜前体细胞能够与其建立细胞连接。这些视网膜前体细胞分化后能够表达胶质纤维酸性蛋白、微管相关蛋白-2和视紫红质，分别为神经胶质细胞、神经元和光感受器细胞的特异蛋白。结论人视网膜前体细胞具有神经干细胞特征，在体外移植到培养的人视网膜组织片下，能够分化成相应的终末分化细胞。

Objective How to modulate the retinal progenitor cells（RSCs） differentiating into special cells is still the hot point. Human RPCs have successfully been isolated. In this study, we observe the differentiation of RPCs after being transplanted to cultured neural retina in vitro. Methods The RPCs were isolated from legally allowed abortion embryonic retina without ocular deformity and were cultured in a selective medium. The RPCs of the second passage were co-cultured with human neural retina for 10 days after being transplanted to subsurface of retina. The cell differentiation and tissue structure were observed by microscope and immunochemistry. Results The RPCs was homogeneously round shape and proliferated and formed sphere-shaped cell clusters. Secondary spheres could be formed in filial generation. All the RPCs spheres expressed Nestin in cytoplasm to form a round brown shape. But the expression of Nestin was negative in mature retinal cells. After being transplanted to retinal tissue, the RPCs could differentiate into various mature cells with intercellular connections. Few of them migrated into the layers of the retinal tissue. Some transplanted RPCs were labeled by bromodeoxyuridine, showing a red fluorescence, and some showed green fluorescence for glial fibrillary acidic protein. The expression of microtubule-associated protein-2 and rhodopsin in transplanted cells were also identified, showing a brown dye. Conclusion Human RPCs manifeste some characterizations of neural stem cells. After being transplanted to neural retina tissue in vitro, they can differentiate into several kinds of mature retinal cells.