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

Effect of melatonin on the proliferation, apoptosis, and expression of bcl-2 in oxidized low-density lipoprotein-induced endothelial progenitor cells

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。

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 iso- lated 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 20 mg/L） for 24 hours ], and 3 Mel groups [ the at- tached cells were incubated with different concentrations of Mel （0.5,1.0, and 2.0 mmol/L） re-spectively 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 -dimethyhhiazol-2 -yl ） -2,5 -diphenyhetrazoli- um bromide （MTI＂） 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 apopto- sis 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 the apoptosis of the cells by down-regulating the bcl-2 ox- LDL. inhibit the proliferation of EPC and promote expression. Mel can inhibit these effects of ox- LDL.