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目的 探讨人胚眼视网膜光感受器细胞的获取和体外培养方法 ,为进行人类视网膜光感受器细胞移植提供理论依据。方法 采用酶分层消化法分离获取人胚眼纯视网膜光感受器细胞 ,使用视网膜色素上皮细胞的条件培养液 ,在体外长期培养并进行形态学鉴别 ;用苏木素 伊红染色显示消化后的视网膜组织结构层次 ,以确定所获取的视网膜光感受器细胞的纯度 ;用抗视杆细胞特异性视紫质蛋白抗体 (rhodopsin4D2 ,Rho4D2 )免疫细胞化学染色做细胞鉴定。结果 酶分层消化法能获取较纯的视网膜光感受器细胞 ,抗Rho 4D2阳性 ,并能在体外存活较长时间。结论 酶分层消化法所获取的人胚眼纯视网膜光感受器细胞能在体外较长时间存活 ,并可表达光感受器细胞特异性视紫质蛋白 ,将为人类视网膜光感受器细胞的移植和各种体外研究提供细胞来源。
Objective To investigate the method of obtaining and culturing retinal photoreceptor cells in human embryo and provide a theoretical basis for human retinal photoreceptor cell transplantation. Methods Human retinal photoreceptor cells were isolated by enzymatic layered digestion and cultured in conditioned media of retinal pigment epithelial cells for long-term culture in vitro. Morphology was identified by hematoxylin and eosin staining of the retina after digestion Level to determine the purity of the retinal photoreceptor cells obtained; cell identification was performed by immunocytochemical staining with anti-rod-specific rhodopsin antibody (rhodopsin4D2, Rho4D2). Results Enzymatic stratified digestion method can obtain pure retinal photoreceptor cells, anti-Rho 4D2 positive, and can survive longer in vitro. Conclusion Enzymatic layered digestion of human retina photoreceptor cells can survive longer in vitro and express photoreceptor cell-specific rhodopsin, which will be useful for human retinal photoreceptor cell transplantation and various In vitro studies provide a source of cells.