NF-κB p65对OX-LDL处理的内皮祖细胞增殖活性的影响及机制研究*

Effect of NF-кB p65 on proliferative activity of OX-LDL treated endothelial progenitor cells *

摘要:

目的 研究核因子κB p65（NF-кB p65）对氧化低密度脂蛋白（OX-LDL）处理的内皮祖细胞增殖活性的影响及机制。方法 分离培养人内皮祖细胞，用OX-LDL细胞培养液培养后，分别用实时荧光聚合酶链反应（qRT-PCR）和Western blotting检测细胞中NF-κBp65 mRNA及蛋白水平。用NF-κB p65 siRNA慢病毒感染内皮祖细胞，给予OX-LDL处理以后，qRT-PCR和Western blotting检测干扰效果。MTT检测内皮祖细胞的增殖活性，流式细胞术检测内皮祖细胞的凋亡变化，Western blotting检测细胞中活化的Caspase-3、Caspase-9蛋白水平，用比色法检测细胞裂解液中的超氧化物歧化酶（SOD）活性及丙二醛（MDA）含量。结果 OX-LDL处理可以诱导内皮祖细胞中NF-κB p65表达上调。NF-κB p65 siRNA慢病毒可以下调OX-LDL条件下内皮祖细胞中NF-кBp65的表达水平。OX-LDL处理后的内皮祖细胞的增殖活性降低，细胞凋亡率升高，细胞中活化的Caspase-3、Caspase-9蛋白水平也升高，细胞中SOD活性降低，MDA含量升高（P?<0.05）。敲低NF-κB p65 后的内皮祖细胞经OX-LDL诱导后，细胞增殖活性有所升高，细胞凋亡率降低，细胞中活化的Caspase-3、Caspase-9蛋白水平降低，SOD活性升高，MDA含量降低（P?<0.05）。结论 敲低NF-κB p65能够降低OX-LDL诱导的氧化应激及细胞凋亡，提高内皮祖细胞增殖活性。

Abstract:

Objective To study the effect and mechanism of NF-кB p65 on proliferation of OX-LDL treated endothelial progenitor cells. Methods Human endothelial progenitor cells was isolation and cultured. After co-culture of OX-LDL with cells, qRT-PCR and Western blotting were used to detect NF-κB p65 mRNA and protein levels in cells. NF-κB p65 siRNA lentivirus infects endothelial progenitor cells. After treated with OX-LDL, qRT-PCR and Western blotting were used to determine the interference effect. MTT assay was used to determine the proliferative activity of endothelial progenitor cells. The apoptosis of endothelial progenitor cells was measured by flow cytometry. Western blotting detected the level of activated Caspase-3 and Caspase-9 protein in the cells. The activity of SOD and the content of MDA in cell lysate were detected by colorimetry. Results OX-LDL treatment induced NF-κB p65 expression in endothelial progenitor cells. NF-κB p65 siRNA downregulated the expression of NF-κB p65 in endothelial progenitor cells under the condition of OX-LDL. OX-LDL treatment induced decrease of proliferation activity and SOD activity while increase of apoptosis, Caspase-3, Caspase-9 and MDA when compared with control group, which were abolished by knock down of NF-κB p65. Conclusions Knocking down of NF-κB p65 reduces oxidative stress and apoptosis induced by OX-LDL, increasing the proliferation activity of endothelial progenitor cells.