人胚胎视网膜来源神经干细胞体外培养尝试  

Isolation and identification of human embryonic retinal stem cells

作　　者：俞海燕[1] 沈丽[2] 王薇[1] 

机构地区：[1]北京大学第三医院眼科中心,100038 [2]北京大学干细胞中心

出　　处：《中华眼科杂志》2005年第9期847-852,共6页Chinese Journal of Ophthalmology

基　　金：国家自然科学基金资助项目(30170999);北京市科委基金资助项目(H020220010390)

摘　　要：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RTPCR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。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 （ Mueller cells） and retina-specific neurons expressing rhedopsin, syntaxin, PKCα, and Thyl. These were markers for red 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. （Chin J Ophthalmol , 2005,41：847-852 ）

关 键 词：胚胎视网膜 神经干细胞 体外培养 细胞分化 

分 类 号：R329.2[医药卫生—人体解剖和组织胚胎学]