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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 investigate the effects of melatonin on proliferation and apoptosis of endothelial progenitor cells (EPCs) induced by oxidized low-density lipoprotein (ox-LDL) Bcl-2 expression. Methods: Human umbilical vein blood mononuclear cells were obtained by density gradient centrifugation. After 7 days of culture, adherent cells were divided into 7 groups: control group was cultured in RPMI1640 culture medium; ox-LDL concentration groups (3 groups) , 10,20 mg / L ox-LDL in RPMI 1640 medium. Each concentration group (3 groups) of Mel was incubated with RPMI 1640 medium containing 0.5, 1.0, 2.0 mmol / L Mel for 24 h, The RPMI 1640 medium containing Mel was incubated with RPMI 1640 medium containing 10 mg / L ox-LDL. The proliferation of EPCs was detected by MTT assay; the apoptosis rate was detected by flow cytometry; RNA was extracted and the expression of Bcl-2 mRNA was detected by reverse transcription-polymerase chain reaction (RT-PCR); Immunocytochemistry Bcl-2 protein expression level. Results: Ox-LDL inhibited EPC proliferation in a dose-dependent manner and induced EPC apoptosis. The addition of Mel before adding ox-LDL (10 mg / L) significantly enhanced EPC proliferation compared with ox-LDL group The expression of Bcl-2 mRNA and protein in EPCs in ox-LDL group was significantly lower than that in control group, and was significantly higher in Mel group than that in ox-LDL group (P <0.01) by RT-PCR and immunocytochemistry. CONCLUSION: Ox-LDL induces EPC apoptosis in a concentration-dependent manner and inhibits the proliferation of EPC. Mel can inhibit the above-mentioned effects of ox-LDL, and its mechanism is related to the up-regulation of Bcl-2 expression.