人胚胎视网膜前体细胞的体外培养条件及分化特性  

In vitro culture and differentiation of fetal human retinal progenitor cells

作　　者：钟秀风[1] 彭福华[2] 葛坚[1] 黄冰[1] 李永平[1] 

机构地区：[1]中山大学中山眼科中心、国家眼科学重点实验室,广东省广州市510060 [2]中山大学附属第三医院神经科,广东省广州市510630

出　　处：《中国组织工程研究与临床康复》2008年第51期10117-10120,共4页Journal of Clinical Rehabilitative Tissue Engineering Research

基　　金：广东省科技计划项目(2006B36006007;2008B030301081)~~

摘　　要：背景：视网膜干细胞已在多种脊椎动物眼内发现，但目前对人类视网膜干细胞或前体细胞的生物学特性尚知之甚少。目的：探讨人胚胎视网膜前体细胞的体外培养条件及分化潜能。设计、时间及地点：细胞学体外观察，于2005-03／2006-02在中山大学中山眼科中心完成。材料：人胚胎神经视网膜来源于4例流产胎儿。方法：分离胚胎眼球神经视网膜，经胰酶消化法制备单细胞悬液，加入含表皮生长因子、碱性成纤维细胞生长因子、白血病抑制因子、左旋谷氨酰胺的无血清高糖DMEM／F12培养基进行原代及传代培养。收集培养第1～3代的细胞克隆球体，接种到右旋多聚赖氨酸及层粘连蛋白预包被的6孔细胞培养板内，培养7d后行免疫细胞化学染色。主要观察指标：倒置显微镜下观察视网膜前体细胞分化前后的形态变化，免疫细胞化学法鉴定分化细胞特性。结果：部分人胚胎神经视网膜细胞呈神经球体样悬浮生长，细胞球体边界清楚，折光性强，表达视网膜前体细胞标签巢蛋白；将其接种到分化条件下培养7d后，细胞体积增大，变扁平，呈梭形、多角形，伸出大小不等的细胞突起，大部分细胞表达视网膜神经元细胞分子标签βⅢtubulin和胶质细胞标签胶质纤维酸性蛋白，少量细胞表达光感受器标签视紫红质。结论：人胚胎视网膜前体细胞可在体外扩增培养，在胎牛血清联合右旋多聚赖氨酸、层粘连蛋白特定诱导条件下，可以向视网膜神经元、胶质细胞及光感受器细胞分化。BACKGROUND： Retinal stem cells or retinal progenitor cells have been found in eyes of several vertebrate animals. However, at present, the biological characteristic of human retinal progenitor cells is still unclear. OBJECTIVE： To explore the culture method, growth and differentiation characteristics of fetal human retinal progenitor cells. DESIGN, TIME AND SETTING： Cytology in vitro study was performed between March 2005 and February 2006 at Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, Guangdong Province, China. MATERIALS： Fetal human retinas were derived from four fetuses by induction of labor. METHODS： Single cell suspension was prepared from fetal human neural retinas with trypsin digestion. Then cells were seeded in serum free chemical defined medium, supplemented with epidermal growth factor, basic fibroblast growth factor, leukaemia inhibitory factor and laevo-glutamine for primary and passage cultures. At first to third passage, cell clone spheres were harvested and incubated in 6-well culture plate coated with dextro-rotatory polylysine and laminin. Seven days after primary culture, immunocytochemical stain was performed 7 days later. MAIN OUTCOME MEASURES： Morphological changes in retinal precursor cells before and after differentiation were observed under inverted microscope. Immunocytochemistry was used to identify characteristics of human retinal progenitor cells before and after differentiation. RESULTS： Some of the fetal human retinal cells proliferated as nonadherent floating neurosphere-like spheres. The cell spheres appeared phase-bright, expressing nestin, a marker for retinal precursors. When they were cultured under differentiation conditions for 7 days, these cells became larger, appeared applanate, fusiform or polygonal in shape with a few processes. Majority of the differentiated cells expressed neural and glial markers β Ⅲ tubulin and glial fibrillary acidcic protein, while minority of them expressed rod cell marker rhodopsin. CONCLUSION： Fetal human

关 键 词：视网膜前体细胞 培养 诱导分化 神经元 

分 类 号：R394.2[医药卫生—医学遗传学]