EC-SOD在同型半胱氨酸致单核细胞源性巨噬细胞氧化应激中的作用研究

发布时间：2018-06-24 12:30

本文选题：细胞外超氧化物岐化酶 + 同型半胱氨酸　；参考：《中国动脉硬化杂志》2017年01期

【摘要】：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(P0.01),EC-SOD mRNA和蛋白表达明显降低(P0.01)。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%(P0.05,P0.01)。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%(P0.05,P0.01)。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。
[Abstract]:Objective to investigate the role and mechanism of extracellular superoxide dismutase (EC-SOD) in oxidative stress of macrophages induced by homocysteine (Hcy). Methods THP-1 mononuclear cells were stimulated with phorbol ester for 48 h and then developed into macrophages. The cells were treated with 0,50100200500 渭 mol / L Hcy for 72 h and treated with Vitamin B12 folate (100 渭 mol / L Hcy 30 渭 mol / L folic acid 30 渭 mol / L vit B12) for 72 h. The expression level of EC-SOD mRNA in macrophages was detected by real-time fluorescence quantitative PCR and the protein expression level of EC-SOD in macrophages was detected by Western blot. The activity of EC-SOD in macrophages was detected by EC-SOD assay kit. EC-SOD recombinant plasmids and interference plasmids were constructed respectively to detect the expression level of EC-SOD mRNA and protein and the expression of superoxide anion. Results compared with the control group, the activity of H _ 2O _ 2 O _ (-)-increased significantly (P0.01) and the expression of EC-SOD mRNA and protein decreased significantly (P0.01) in the 100200 渭 mol / L Hcy group. Compared with the control group, the EC-SOD activity in the 100200500 渭 mol / L Hcy group decreased by 13.92% (P0.05P0.01), and 8.62 渭 mol / L Hcy group decreased the EC-SOD activity by 10.32% (P0.05P0.01), respectively. Compared with 100 渭 mol / L Hcy group, the expression of EC-SOD mRNA in Vit B12 intervention group increased 47%. After transfection of EC-SOD recombinant plasmid and interference plasmid, compared with 100 渭 mol / L Hcy group, the content of O _ 2O _ 2 in EC-SOD recombinant group decreased 63.89%, and the content of O _ 2O _ (2) in interference fragment -596 group increased 33.59% (P 0.05 ~ (0.05) P _ (0.01). Conclusion EC-SOD is involved in the oxidative stress of monocyte derived macrophages induced by Hcy. EC-SOD may play an important role in inhibiting atherosclerosis induced by Hcy.
【作者单位】：宁夏医科大学基础医学院;宁夏医科大学药学院;
【基金】：国家自然科学基金项目(81360052、81560084、81460080) 2014年度校级优势学科群项目(XY201415)
【分类号】：R543.5

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