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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 culture neural stem cells from human embryonic retina. Methods The single-cell suspensions prepared from the retina of human embryos from 16 to 20 weeks were cultured in vitro in serum-free and serum-conditioned medium. The expression of nestin and retinal terminal-cell antigen in cultured cells was detected by immunofluorescence. The RTPCR method And real-time fluorescent quantitative PCR method to detect the difference of nestin gene mRNA expression before and after induction. As a result, neural stem cells can be cultured from the human embryonic retinal nerve sensory layer. The cells were self-replicating and expressing nestin. After induced by different conditions, the cells expressed markers such as retinal rod cells, amacrine cells, bipolar cells, ganglion cells and Miller cells. After induction, the expression of nestin gene was significantly lower than that before induction. Conclusion There are self-renewal and multi-differentiation neural stem cells in human embryonic retinal nerve sensory layer.