AIM:To investigate the cobalt chloride(CoCl2)simulated hypoxic environment's effect on the gene expression and protein secretion of vascular endothelial growth factor(VEGF)of rats' retinal progenitor cells(RPCs). 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.

Background In vitro study showed that chemotaxis consist of chemotaxis factor 4(CXCR4)and stromal cells derived factor-1(SDF-1)and may play a role in the orientation and migration of retinal progenitor cells (RPCs)toward lesion.Overexpression of CXCR4 in RPCs can enhance the chemotaxis activity.Objective This work was to explore the feasibility and underlying mechanism of up-regulation of CXCR4 on RPCs induced by hypoxia.Methods RPCs were retained in an incubator with normal O2volume(16％)or hypoxia condition(10％ O2)for 12 hours and 24 hours respectively.Flow cytometer cell analysis screening(FACS)was conduced to measure the proportion of CXCR4-expressing cells,and CXCR4,HIF-1 mRNA were analyzed by reverse transcription-polymerse chain reaction(RT-PCR).The chemotical effect of 30 mg/L SDF-1 to RPCs cultured under the hypoxia condition was assessed using Boyden chamber.Results The expression level of CXCR4(CXCR4 mRNA/β-actin mRNA)inRPCs cultured by 10％ O2 for 12 and 24 hours were 0.28+0.07and 0.48+0.17 and increased by 1.75 and 3.00 fold more than that of 16％ O2 culture group(0.16+0.02)(P＜0.01).The expression level of HIF-1 mRNA(HIF-1 mRNA/β-actin mRNA)in RPCs cultured by 10％ O2 for 12 and 24 hours were 0.18 ±0.07and 0.38 ±0.13 and increased by 3.00 and 6.30 fold more than that of 16％ O2 culture group(0.06±0.01)(P＜0.01).The chemotical effect of 30 μg/L SDF-1 to RPCs increased from 13.00％ in 16％ O2 culture group to 36.00％ and 46.00％ in the cells cultured by 10％ O2for 12 and 24 hours.FACS revealed that the proportion of CXCR4+ cells in hypoxia-exposure for 12 and 24 hours were 26.90％ and 46.10％,respectively,but that in 16％ O2 culture group was 9.10％,showing a statistically significant difference(P ＜ 0.01).Conclusions RPCs induced by hypoxia can enhance the expression of CXCR4 in RPE cells and the chemotaxia to SDF-1.The overexpression of H1F-1 in RPCs may be involved in the up-regulation of CXCR4 expression.