方法：全神经球贴壁法分离、培养、鉴定RPCs。应用RT-PCR及ELISA，检测不同浓度 CoCl₂(0，50，100，150，200μmol/L)干预24h后RPCs中VEGF表达的变化，检测150μmol/L CoCl₂作用不同时间(0，12，24，36，48，72h)RPCs中VEGF表达的变化。

结果：全神经球贴壁法培养的RPCs高表达神经干细胞特异性标记(Nestin)，自然分化后表达视网膜细胞标志性抗原Rhodopsine，MAP-2和GFAP，在缺氧培养时间相同的情况下，VEGF 基因的表达随着 CoCl₂浓度的增加而逐渐增加而后降低，CoCl₂为150μmol/L时达到峰值(P≤0.05)。当 CoCl₂浓度为150μmol/L培养不同时间时，随着缺氧干预时间的延长，VEGF基因的表达亦是逐渐增加而后降低，于36h时达到峰值(P≤0.05)，蛋白与基因表达的变化基本一致。

结论：全神经球贴壁法培养RPCs能较好地表达其干细胞特性。CoCl₂模拟缺氧环境下，缺氧使 RPCs中VEGF 的分泌增强并具有时间和剂量依赖性。

METHODS: RPCs were separated, cultured and identified by the use of neurospheres-adhesive culture. RT-PCR and ELISA were used to detect the change of RPCs VEGF's expression after 24-hour intervention of CoCl₂ with different concentration(0, 50, 100, 150μmol/L and 200μmol/L), and detect the change of RPCs VEGF's expression in 150μmol/L CoCl₂ for different period(0, 12, 24, 36, 48, 72 hours).

RESULTS: The RPCs cultured by neurospheres-adhesive culture system could highly express the specificity of stem cells(Nestin), and express mature retinal neuron after natural differentiation. After culturing them in the hypoxic environment for the same period, the gene express of VEGF gradually increased and then decreased with the increasing concentration of CoCl₂, and peaked(P≤0.05)when the concentration of CoCl₂ was 150μmol/L. When the concentration of CoCl₂ was 150μmol/L, the gene expression of VEGF also gradually increased and then decreased with the increasing hypoxia intervention period, and peaked(P≤0.05)when the hypoxia intervention period was 36 hours, the change of protein and gene expression were consistent.

CONCLUSION: The RPCs cultured by neurospheres-adhesive culture system can better express the specificity of stem cells. Under CoCl₂ simulated hypoxic environment, hypoxia strengthens RPCs VEGF's secretion and dependent on the time and dose.