Effects of recombinant adeno-associated virus on proliferation and apoptosis of endothelial cells

Objective To investigate the effects of type 9 recombinant adeno-associated virus (rAAV9) mediated anti-nuclear factor-κB (NF-κB) ribozyme on the proliferation and apoptosis of high lipid serum-induced human umbilical vein endothelial cells (HUVECs) in vitro,and to provide an important support that R65 nuclear enzyme gene would be an optimal gene for gene therapy in AS.Methods HUVECs were cultured in vitro,endothelial damage model of AS was established by using high lipid serum,and the HUVECs were transfected by the rAAV9-R65 recombinant adenovirus vector.Cells were divided into three experimental groups:control group,high lipid serum induced group and high lipid serum induced + rAAV9-R65 transfected group.The expression of p65 protein was detected by using Western blotting.The cell supernatant fluid was collected for determination of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) by using enzyme linked immunosorbent assay (ELISA).Proliferation of HUVECs transfected by rAAV9-R65 was detected by using Alamar blue.The apoptosis rate was detected by using flow cytometry 96 h after transfection by rAAV9-R65.Results The cells were changed from long spindle into class circle after 24 h induced by high lipid serum.Western blotting showed that the expression of p65 in control group was 0.022 9 ± 0.002 4 ; In high lipid serum induced group was 0.224 3 ± 0.008 6 ; In rAAV-R65 transfected and high lipid serum induced group was 0.071 4 ±0.007 0.The expression of p65 in control group was increased after high lipid serum stimulating HUVECs,and decreased statistically significant in rAAV-R65 transfected and high lipid serum induced group.ELISA showed that the concentrations of TNF-α in the supernatant among three groups were:15.98 ±0.43,27.94 ± 0.83 and 19.73 ± 0.43,respectively.And the concentrations of IL-6 among three groups were:3.94 ±0.12,6.32 ± 0.21 and 5.08 ± 0.12,respectively.The levels of TNF-α and IL-6 were significantly higher than control group after 24 h induced by high lipid serum,while they were decreased significantly in rAAV-R65 transfected and high lipid serum induced group.Alamar blue showed that the expression of R65 gene could significantly reduce the proliferation inhibition of HUVECs after high lipid serum stimulation.Flow cytometry analysis showed that the early apoptosis rate in high lipid serum induced group was 20.4％,and the late apoptosis ratio was 4.7％ compared with the control group.While in rAAV-R65 transfected and high lipid serum induced group it was 7.2％ of early apoptosis rate and late apoptosis ratio was 2.9％.It showed that the apoptosis rate of HUVECs was decreased by R65 gene.Conclusion The expression of R65 gene could decrease the proliferation inhibition of HUVECs after the high lipid serum induction,and could inhibit the apoptosis.These findings carry significant implications for adenovirus mediated R65 for gene therapy of AS. Key words: R65 nuclear enzyme gene;  Adeno-associated virus;  High lipid serum;  Proliferation ;  Apoptosis