美乐托宁对ox-LDL诱导的人脐静脉血内皮祖细胞增殖、凋亡及Bcl-2表达的影响

目的:探讨美乐托宁(melatonin,Mel)对氧化低密度脂蛋白(oxidized low-density lipoprotein,ox-LDL)诱导的人脐静脉血内皮祖细胞(endothelial progenitor cell,EPC)增殖、凋亡及Bcl-2表达的影响.方法:密度梯度离心法获取人脐静脉血单个核细胞,培养7 d后,贴壁细胞分成7组:对照组以RPMI1640培养液培养;ox-LDL各浓度组(共3组)分别用含5,10,20 mg/L ox-LDL的RPMI 1640培养液孵育;Mel各浓度组(共3组)分别先用含0.5,1.0,2.0 mmol/L Mel的RPMI 1640培养液培养24h,然后移去含Mel的RPMI 1640培养液,加入含10 mg/L ox-LDL的RPMI 1640培养液孵育.采用MTT法检测EPC增殖能力;流式细胞仪检测细胞凋亡率;提取细胞RNA,采用逆转录一聚合酶链式反应(RT-PCR)技术检测Bcl-2 mRNA表达;采用免疫细胞化学法检测Bcl-2蛋白表达水平.结果:ox-LDL呈浓度依赖性抑制EPC增殖,诱导EPC凋亡;在加ox-LDL(10 mg/L)前加Mel干预,EPC增殖能力较ox-LDL组明显增强,细胞凋亡率明显降低;RT-PCR和免疫细胞化学法检测显示,ox-LDL组EPC的Bcl-2 mRNA和蛋白表达明显低于对照组,Mel组明显高于ox-LDL组(P＜0.01).结论:ox-LDL呈浓度依赖性诱导EPC凋亡、抑制EPC增殖,Mel能抑制ox-LDL的上述作用,其机制与上调Bcl-2表达有关.

Effect of melatonin on the proliferation, apoptosis, andexpression of bcl-2 in oxidized low-densitylipoprotein-induced endothelial progenitor cells

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 groupsthe attached cells were incubated with different concentrations of ox-LDL(5,10,and 20 mg/L) for 24 hours], and 3 Mel groupsthe 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.