人胚胎视网膜来源神经干细胞体外培养尝试

目的从人胚胎视网膜中分离、培养神经干细胞。方法人16～20周胚胎视网膜制备的单细胞悬液,分别在无血清和有血清条件下进行体外培养,采用免疫荧光法检测培养的细胞对nestin和视网膜终末细胞抗原的表达,采用RT—PCR方法和实时荧光定量PCR法检测诱导前后细胞nestin基因在mRNA水平的表达差异。结果可从人胚胎视网膜神经感觉层中培养出神经干细胞。该细胞具有自我复制能力,表达nestin,经不同条件诱导后细胞表达视网膜视杆细胞、无长突细胞、双极细胞、节细胞和米勒细胞等标志。诱导后细胞nestin基因表达量较诱导前细胞明显降低。结论人胚胎视网膜神经感觉层中存在具有自我更新能力和多分化潜能的神经干细胞。（中华眼科杂志,2005,41：847-852）

Isolation and identification of human embryonic retinal stem cells

OBJECTIVE: To isolate and identify human retinal stem cells (HRCs) from human embryonic retina. METHODS: HRCs were isolated from neural retinas at the 16th-20th week of gestation and cultured in either serum-free or serum-containing media. The expression of nestin antigen and retinal terminal cell markers were conformed by immunofluorescence labeling technique. RT-PCR analysis was used to identify the difference of nestin gene expression in HRCs and differentiated cells. RESULTS: HRCs divided into multiple generations (up to passage 8) and expressed nestin. Other characteristics of the isolated cells were to differentiate into glial fibrillary acidic protein-positive glial cells (Müller cells) and retina-specific neurons expressing rhodopsin, syntaxin, PKCalpha, and Thy1. These were markers for rod receptors, amacrines, bipolar cells, and ganglion cells respectively. When cultured with serum, the expression of nestin was significantly downregulated in HRCs. CONCLUSION: Human neural retina at the 16th-20th week of gestation harbors neural stem cells that are capable of selfrenewal and multilineage differentiation.