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目的探讨细胞外超氧化物岐化酶(EC-SOD)在同型半胱氨酸(Hcy)致巨噬细胞氧化应激中的作用及机制。方法将THP-1单核细胞用佛波酯刺激48 h后演变成巨噬细胞,用0、50、100、200、500μmol/L Hcy作用细胞72 h,并加设叶酸+维生素B12(Vit B12)干预组(100μmol/L Hcy+30μmol/L叶酸+30μmol/L Vit B12)。微板法检测氧化应激指标(H_2O_2、O_2~-、OH-)的变化;实时荧光定量PCR检测巨噬细胞中EC-SOD的mRNA表达水平;Western blot检测巨噬细胞中EC-SOD的蛋白表达水平;EC-SOD测定试剂盒检测EC-SOD活性。分别构建EC-SOD重组质粒和干扰质粒转染细胞,检测EC-SOD的mRNA及蛋白的表达水平以及超氧阴离子的表达。结果与对照组相比,100、200、500μmol/L Hcy组H_2O_2、OH-活性显著增高(P<0.01),EC-SOD mRNA和蛋白表达明显降低(P<0.01)。与对照组相比,100、200、500μmol/L Hcy组EC-SOD活性分别下降了13.92%、8.62%、10.32%(P<0.05,P<0.01)。与100μmol/L Hcy组相比,叶酸+Vit B12干预组EC-SOD mRNA的表达升高了47%。分别转染EC-SOD重组质粒和干扰质粒后,与100μmol/L Hcy组相比,EC-SOD重组组O_2~-含量降低了63.89%,干扰片段-596组O_2~-含量则增加了33.59%(P<0.05,P<0.01)。结论 EC-SOD参与了Hcy导致的单核细胞源性巨噬细胞的氧化应激。在抑制Hcy诱导动脉粥样硬化的过程中,EC-SOD可能发挥着重要的作用。
Objective To investigate the role and mechanism of extracellular superoxide dismutase (EC-SOD) in oxidative stress induced by homocysteine (Hcy) in macrophages. Methods THP-1 monocytes were stimulated with phorbol ester for 48 h and then transformed into macrophages. The cells were treated with 0, 50, 100, 200 and 500 μmol / L Hcy for 72 h, and folic acid plus vitamin B12 (Vit B12) ) Intervention group (100μmol / L Hcy + 30μmol / L folic acid + 30μmol / L Vit B12). The changes of oxidative stress index (H_2O_2, O_2 ~ -, OH_) were detected by microplate method. The mRNA expression of EC-SOD in macrophages was detected by real-time fluorescence quantitative PCR. The protein of EC-SOD in macrophages The expression level of EC-SOD was detected by EC-SOD assay kit. The recombinant plasmids of EC-SOD and plasmids were constructed respectively to detect the expression of EC-SOD mRNA and protein and the expression of superoxide anion. Results Compared with the control group, the activities of H 2 O 2 and OH- in 100, 200 and 500 μmol / L Hcy groups were significantly increased (P <0.01) and the expressions of EC-SOD mRNA and protein were significantly decreased (P <0.01). Compared with the control group, the activity of EC-SOD decreased by 13.92%, 8.62% and 10.32% (P <0.05, P <0.01) in 100, 200 and 500μmol / L Hcy groups, respectively. Compared with the 100μmol / L Hcy group, the expression of EC-SOD mRNA increased by 47% in the folic acid + Vit B12 intervention group. Compared with 100 μmol / L Hcy group, the content of O 2 - in EC-SOD recombinant group decreased 63.89% and the content of O 2 - in interference fragment-596 group increased 33.59% after transfection with EC-SOD recombinant plasmids and interference plasmids, respectively (P <0.05, P <0.01). Conclusion EC-SOD is involved in Hcy-induced oxidative stress in monocyte-derived macrophages. EC-SOD may play an important role in inhibiting Hcy-induced atherosclerosis.