Background Retinal hypoxia is one of primary causes of retinal neovascularization,and its mechanism is research hot topic.Studies showed that hypoxia stimulates the activation of many trancripation factors and vascular endothelial cells,which leads to angiogenesis.Besides to the vascular endothelial growth factor,the effect of adenosine on angiogenesis is increasingly concerned.Objective This study was to invastigate the relationship of biological behaviour of human microvascular endothelial cells (HMEC-1) under the hypoxia and adenosine,and to explore the effect of adenosine on angiogenesis under hypoxia.Methods HMEC-1 cell line was cultured in vitro,and the cells were divided into normoxia group and hypoxia group.The cells in the normoxia group were cultured under the 5％ CO2 environment,and those in the hypoxia group were cultured under the 1％ O2,94％ N2 and 5％ CO2 environment.The proliferation ability and percentage of the cells were assayed by cell counting kit-8 (CCK8) and EDU.The migration number and invasive number of the cells were detected by transwell chamber.The expressions of CD39 and CD73 proteins in the cells were tested by Western blot and immunofluorescence technique,and the level of adenosine was measured by high performance liquid chromatography (HPLC).Results The proliferation values (absobancy) were 0.715-±0.067 and 0.821 ±0.056 in the normoxia group and the hypoxia group in 12 hours after culture,and those in 24 hours were 0.946±0.028 and 0.998±0.028,showing significant increase in the hypoxia group compared with the normoxia group (t12h =3.805,t24h =3.222,all at P ＜ 0.01).The precentage of the proliferation in the hypoxia group was evidently higher than that in the normoxia group (t =-6.868,P＜0.01).The number of the cell migration and invasion in 24 hours after culture was 185.3 ± 10.594 and 74.2± 10.741 respectively in the normoxia group,and that in the hypoxia group was 300.7±22.853 and 107.5±7.007,with significant differences between the two groups (t=-12.124,-6.367,both at P＜0.01).The expression levels of CD39 and CD73 proteins in the hypoxia group were significantly higher than those in the normoxia group in bothl2 hours and 24 hours after culture(all at P＜0.05),and the adenosine content in the cells is gradually increased in 2,6,12,24 and 36 hours after culture,with the highest content in 36 hours.The adenosine content was significantly higher in the hypoxia group than in the normoxia group at various time points (t2h =2.469,P =0.017;t6h =5.442,P＜0.001;t12h =3.841,P＜0.001;t24h =4.458,P＜0.001;t36h =2.757,P =0.008;t48h =3.319,P =0.002).Conclusions Compared with the normoxia group,the proliferation,migration and invasion abilities of HMEC-1 are stronger,meanwhile,the expression of CD39 and CD73 as well as the adenosine level in the cells are all increased under the hypoxic condition.It is suggested that the activation of human microvascular endothelial cells might be significantly related to the level of adenosine and its key enzymes.

Background Retinal ganglion cell (RGCs) death following ischaemic insult is the major cause of a number of vision-threatening diseases.Recent studies confirmed that micro RNA (miR-30b) can alleviate hypoxy-induced cardiac injury.However,whether miR-30b can protect RGCs against oxygen-glucose deprivation damage is still not ellucidated.Objective The aim of this study was to investigate the protective effect of miR-30b on RGCs damage caused by oxygen-glucose deprivation.Methods The retinas were isolated from the eyeballs of eight SD rats aged postnatal 24 hours and RGCs were primarily cultured.The cells were divided into the recombinant adeno-associated virus (rAVV) control group,rAAV-miR-30b mimic group and AAV-miR-30b inhibitor group.Then the cells were transfected using rAVV-miR plasmid,rAAV-miR-30b mimic plasmid and AAV-miR-30b inhibitor plasmid,respectively for 6 days with the RGCs ∶ AAV as 1 ∶ 10 000.The cells were cultured with low glucose medium in hypoxygen incubator (5％ CO2,17％ N2,3％ O2) or 5％ CO2 incubator respectively for 24 hours.Cell viability was detected by cell counting kit-8 assay.The expression of Tubulin Ⅲ,a neuron specific marker,was detected by immunofluorescence technology to evaluate the survival of RGCs.The apoptosis and necrosis of the cells were assessed by Hoechst/PI double staining.Results The RGCs grew well with round shape and 1 3 processes 7 days after cultured in the normal cells.However,the RGCs were diminished and the cell process disrupted in the oxygen-glucose deprivation group.The relative vability of the cells was 3.310-±0.162 in the rAAV-miR-30b mimic group,which was significantly higher than 0.949±0.141 in the rAAV-miR-30b inhibitor group and 0.900±0.181 in the rAAV-miR control group(t=10.508,10.296,both at P＜0.001).It was positively expressed in survival RGCs,with the red fluorescence.The number of Tubulin Ⅲ+ cells was (13.800± 1.924)/field in the rAAV-miR-30b mimic group,showing a significant increase in comparison with (0.600±0.548)/field in the rAAV-miR-30b inhibitor group and (0.800± 1.304)/field in the rAAV-miR control group (t =15.141,14.912,both at P ＜ 0.001).Significant differences were found in the apoptosis rate and necrosis rate among the rAAV-miR-30b mimic group,rAAV-miR control group and PBS group (F=10.851,P=0.002;F=6.378,P=0.013),and the apoptosis rate and necrosis rate in the rAAV-miR-30b mimic group were considerably lower than those in the rAAV-miR control group and PBS group (all at P＜0.05).Conclusions The oxygen-glucose deprivation models can be established in RGCs by hypooxygic and low-glucose cultivation.rAAV encoding miR-30b mimics transfection can protect RGCs against oxygen-glucose deprivation damage.