[Establishment of senescent cell model in primary rat aortic endothelial cells]

Objective To establish senescent models in rat aortic endothelial cells (RAECs) induced by high glucose (HG), angiotensin II (AngII), hydrogen peroxide and palmitic acid (PA), and compare the senescence-induced effects of these factors. Methods Primary RAECs were extracted from two-month-old male Wistar rats by issue explant method and identified by CD31 immunofluorescence cytochemistry. RAECs were treated separately by 30 mmol/L (HG), 10 μmol/L AngII, 100 umol/L hydrogen peroxide (H₂O₂) and 0.5 mmol/L PA. Twenty-four hours later, senescence-associated β-galactosidase (SA-β-gal) staining was used to evaluate the senescent state. Real-time quantitative PCR was used to investigate mRNA expression level of senescence-related gene P16. Western blot analysis was performed to determine protein expression levels of P16, P21 and P53. Immunofluorescence cytochemistry was used to detect the expression of P16 protein in cells. The cell viability of RAECs was tested via CCK-8 assay. Results Compared with the control group, positive rate of SA-β-gal staining in each treatment group increased, especially in H₂O₂ and PA groups. And mRNA expression level of P16 increased in all four groups. P16 and P21 proteins had high expression in AngII, H₂O₂ and PA groups, most obviously in H₂O₂ group. P16 immunofluorescence expression level was enhanced in all groups. The cell viability in HG and AngII groups was similar with the control group, while H₂O₂ and PA groups had low cell viability. Conclusion The aging mode of RAECs is successfully established by HG, AngII, H₂O₂ or PA treatment, and H₂O₂ treatment shows the strongest effect.