ABSTRACT
Background Retinal neovascularization disease is a common cause of blinding.Retinal neovascularization is related to enhancing proliferation of vascular endothelial cells.So how to inhibit proliferation of vascular endothelial cells is a hot burning issue.p21 is known to be involved in the regulation of cell cycle and therefore inhibit the cell proliferation.However,the relationship of p21 and the proliferation of vascular endothelial cells in retinal neovascularization disease is for further study.Objective The aim of this experiment was to study the proliferation of rhesus retinal vascular endothelial cells(RF/6A) and expression of p21 in RF/6A cells under the hypoxia condition,and discuss their association.Methods The RF/6A cells were cultured and passaged in vitro,then they were randomly divided into normoxia culture group(5％ CO2 +95％ O2) and hypoxia for 1 hour,3,6,12 hours group(1％ 02+5％ CO2 +94％ N2).Flow cytometer(FCM) was used to check the distribution of RF/6A cell cycle in the normoxia culture group and hypoxia for 1 hour,3,6,12 hours groups.MTT assay was used to detect and compare the cell proliferation(A570)among the various groups.The expression of p21 in the cells was analyzed by Western blot.Results FCM showed that the cells proportion of G0/G1 stage was reduced initially and then increased afterward in hypoxia for 1 hour and 3,6,12 hours groups,showing a significant difference among 5 groups (F =20.083,P =0.000),and the cells proportion of S stage and G2/M stage were increased firstly and then declined in different hypoxia groups with statistical significances (F =7.861,P =0.001 ; F =10.305,P =0.003).Compared with normoxia culture group,cells proportion of G0/G1 stage was declined and that of S stage and G2/M stage were raised after hypoxia culture,showing statistically signifcant differences(P＜0.05).MTT showed that cell multiplication capacity(A570 value)strengthened firstly and then weakened in hypoxia groups with time prolongation,showing a significant difference among all the groups(F=7.768,P=0.001),and A570 value in hypoxia for 3 hours and 6 hours groups (0.315± 0.062,0.365 ± 0.064) was significantly higher than that of the normoxia group (0.205 ± 0.063),respectively(P＜0.05).Western blot showed that the expression of p21 in the cells down-regulated at the beginning and then up-regulated with the increase of hypoxia time,and there was statistical significance (F =16.738,P=0.000).The p21 relative levels in different hypoxia groups were reduced in comparison with the normoxia group,showing statistical signifcances(P＜0.05).Conclusions Short-term hypoxia could reduce the expression of the p21 in RF/6A and induce cell proliferation initially,then p21 increases and cell proliferation is inhibited with the prolongation of hypoxia time.