Objective: To explore the effect of melatonin(Mel) on the proliferation, apoptosis and expression of bcl-2 in oxidized low-density lipoprotein(ox-LDL)-induced endothelial progenitor cells (EPC) from human umbilical cord blood in vitro.
Methods: Total mononuclear cells were isolated from human umbilical cord blood in vitro by Ficoll density gradient centrifugation, and the cells were plated on fibronectin-coated culture dishes. After 7 days, the attached cells were divided into 7 groups: a control group (normal cells), 3 ox-LDL groups[the attached cells were incubated with different concentrations of ox-LDL(5,10,and 20mg/L) for 24 hours], and 3 Mel groups[the attached cells were incubated with different concentrations of Mel (0.5,1.0, and 2.0 mmol/L) respectively for 24 hours before incubation with 10 mg/L ox-LDL]. EPC was identified by examining the expression of CD34, vascular endothelial growth factor receptor-2(VEGFR-2) and CD133 under a laser scanning confocal microscope. We used 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay to detect the effect of Mel and ox-LDL on the multiplication ability of EPC. Flow cytometry was used to detect the apoptosis. The expressions of Bcl-2 mRNA and protein were detected respectively by RT-PCR and immunohistochemistry technology.
Results: After being exposed to the ox-LDL, the proliferation of EPC in the 3 ox-LDL groups was lower, and the apoptosis rate was higher than that in the control group in a dose-dependent manner (P<0.01); Mel was added at different concentrations before the ox-LDL incubation, and the cells in the 3 Mel groups showed higher proliferation and lower apoptosis rate than those of the 3 ox-LDL groups (P<0.01). Expression of Bcl-2 mRNA and protein of EPC in the 3 Mel groups was higher than that in the 3 ox-LDL groups (P<0.01).
Conclusion: Ox-LDL can inhibit the proliferation of EPC and promote the apoptosis of the cells by down-regulating the bcl-2 expression. Mel can inhibit these effects of ox-LDL.