ABSTRACT
Objective To observe the influences of uncoupling protein 2 (UCP-2) rs660339 variants transfection on cell proliferation and apoptosis of human umbilical vein endothelial cell (HUVEC). Methods Two UCP-2 green fluorescent protein (GFP) lentivirus constructs were created with the rs660339 locus carried C or T (UCP-2C or UCP-2T), respectively. HUVEC were cultured after lentiviral infection of UCP-2C or UCP-2T. The expression of UCP-2C or UCP-2T was detected with real time polymerase chain reaction. Cell proliferation and cell apoptosis were compared among negative control (NC) group, UCP-2T group and UCP-2C group using CCK-8 cell viability and flow cytometry. Western blot and immunostaining were employed to examine the expression of Bcl-2 gene. Results The lentivirus constructs were successfully created.>80%of the transfected cells were found to express GFP under fluorescent microscope. The mRNA levels of UCP-2 gene were significantly increased (F=29.183, P=0.001) in the UCP-2T group and UCP-2C group. The CCK-8 assay revealed that on day two (F=15.970, P=0.004), day three (F=16.738, P=0.004), day four (F=5.414, P=0.045) post-infection, UCP-2T and UCP-2C group showed significantly greater proliferation than the NC cells. The apoptotic rate in the UCP-2T and UCP-2C group was significantly lower than NC group (F=277.138, P=0.000), and the apoptotic rate of UCP-2T was significantly lower than that of UCP-2C (P=0.003). The protein levels of Bcl-2 in the UCP-2T and UCP-2C group were significantly greater than that in the NC group (F=425.679, P=0.000), and the Bcl-2 expression of UCP-2T was greater than that of UCP-2C (P=0.002). The Bcl-2 density in the UCP-2T and UCP-2C group were greater than that in the NC group (F=11.827, P=0.008), while there was no difference between UCP-2T and UCP-2C group (P=0.404). Conclusion The variants of UCP-2 rs660339 may influence HUVEC proliferation and apoptosis, and UCP-2T showed a stronger effect of inhibiting apoptosis than UCP-2C.
ABSTRACT
Objective To observe the expression of vascular endothelial growth factor receptor-1 (VEGFR-1) and VEGFR-2 in hypoxic chorioretinal endothelial cells of monkeys (RF/6A),and to evaluate the effect of minocycline.Methods RF/6A was cultured and divided into four groups:control group,hypoxia group,hypoxia and low dose of minocycline group (0.5 μmol/L),hypoxia and medium dose of minocycline group (5 μmol/L),and hypoxia and high dose of minocycline group (50 μmol/L).Real-time reverse transcription-polymerase chain reaction (RT-PCR) and immunohistopathological staining were used to measure the mRNA and protein expression of VEGFR-1 and VEGFR-2,respectively.Results RT-PCR showed that the expression of VEGFR-1 mRNA did not vary significantly between groups (F24 h =0.17,F48 h =1.53,F72 h =2.04; P＞0.05).Compared with hypoxia group,the expression of VEGFR-2 mRNA in all minocycline treated groups were significantly down-regulated (low minocycline,medium minocycline,high minocycline:t=4.69,20.16,17.12; P＜0.001).The immunohistopathological study showed the cells with positive staining of VEGFR-1 can be observed in all groups,and the staining was relatively weak and mainly located in cell membrane and cytoplasm.The optical density value analysis showed that the protein expression of VEGFR-1 did not vary significantly between groups at all time points(F24 h =0.251,F48 h=0.340,F72 h =0.589; P＞0.05).The VEGFR-2 positive staining cells were also observed in all groups,and the staining was relatively high.Brown staining particles of VEGFR-2 were observed in the cell membrane with minor staining particles in cytoplasm.The staining density of VEGFR-2 was significantly higher in hypoxia group than control group.Compared with the hypoxia group,the protein expression of VEGFR 2 in minocycline treated groups was significantly lower (F24 h =19.147,F48 h =14.893,F72 h =11.984; P＜0.05).Conclusion The expression of VEGFR-2 is up-regulated in RF/6A,and minocycline somewhat shows an inhibition effect.