Differences in gene expression profiles of hypoxic human embryonic retinal microvascular endothelial cells induced by cobalt chloride

Abstract

Objective To study the differentially expressed genes and analyze its functional pathways of retinopathy of prematurity(ROP), in order to discover the pathogenesis and provide the theoretical basis for the prevention and treatment of ROP. Methods Fetal eyeballs of induced labor were obtained and retinal microvascular endothelial cells were isolated and cultured.The endothelial cells were divided into 7 groups according to the medium of cobalt chloride (CoCl2) concentration (0 μmol/L, 100 μmol/L, 150 μmol/L, 200 μmol/L, 250 μmol/L, 300 μmol/L, 350 μmol/L), and 150 μmol/L CoCl2 was finally used to induce ROP model in vitro.Retinal microvascular endothelial cells were verified by adopting Ⅷ factor and CD31 antibody fluorescence staining.RNA purification, gene chip hybridization and signi-ficant analysis of microarrays were performed to screen differentially expressed genes.Genes functional pathways were studied by using gene ontology analysis software. Results (1) The proliferation activity of vascular endothelial cells decreased when CoCl2≥150 μmol/L(F=21, P<0.05). (2) In 150 μmol/L CoCl2 group, blue nucleus and green cytoplasm were visible in the second and the third generation vascular endothelial cells stained by factor Ⅷ antibody, and red fluorescence could also be observed in the cytoplasm by means of CD31 monoclonal antibody staining.However, only blue nucleus was detected in the group without CoCl2.(3)There were 326 genes differently expressed in retinal micro vascular endothelial cells induced by CoCl2in vitro, among whom, 198 genes were up-regulated and 128 genes were down-regulated.Up-regulated expression genes were 1.5 times more than those of the down-regulated genes.(4)Ten biological pathways including cell hypoxia, angiogenesis suppression and iron ion transport etc.may play important roles in ROP pathogenesis. Conclusions Hypoxic retinal microvascular endothelial cells induced by CoCl2 can successfully be used to induce cell model of ROP in vitro.Differentially expressed genes may play an important role in ROP development.Functional pathway such as hypoxic cells, inhibition of angiogenesis, iron ion transport may be associated with ROP pathogenesis. Key words: Cobalt chloride; Retinopathy of prematurity; Gene expression profile