Ox-LDL对血管内皮细胞ABCA1及炎症因子表达的研究

Influence of oxidized high density lipoprotein on expression of ATP-binding cassette A1 and cytokines in vascular endothelial cell

目的:研究在致动脉粥样硬化(As)因子氧化低密度脂蛋白胆固醇(Ox-LDL)刺激下,人血管内皮细胞ECV304中ATP-结合盒转运子A1(ABCA1)对炎性细胞因子ICAM-1及IL-1β水平调节作用,阐明ABCA1基因在AS发生中的可能机制。方法:培养人血管内皮细胞株ECV304,加入Ox-LDL(30 ng/ml)刺激3、6、12、24 h,以荧光定量逆转录-聚合酶链式反应(RT-PCR)检测ABCA1、ICAM1 mRNA表达量,Western blot和酶联免疫吸附(ELISA)法检测ABCA1、ICAM1及IL-1β蛋白表达量;ABCA1的反义寡核苷酸(100 nmol/L)转染人血管内皮细胞,按相同方法给予Ox-LDL刺激细胞,同样的方法测定上述指标的改变。阴性对照为未加Ox-LDL刺激并同时培养的细胞。结果:与阴性对照组比较,人血管内皮细胞株ECV304在给予Ox-LDL刺激不同时间后,AB-CA1、ICAM-1的mRNA和蛋白及IL-1β蛋白水平均增高;给予ABCA1反义寡核苷酸转染后各时间点ABCA1、ICAM-1mRNA表达均降低,ABCA1、ICAM-l及IL-1β蛋白表达水平也降低。结论:人管内皮细胞在Ox-LDL作用下,其ABCA1可能通过增加IL-1β的表达,促进炎症细胞因子ICAM-1释放,在AS的早期发生中发挥作用。

Objective：To investigate the modulatory effects of ATP-binding cassette A1 （ABCA1） on intercellular adhension molecule -1 （ICAM-1）, interleukin -1β （IL-1β） levels in vascular endothelial cell after the stimulation with oxidized high density Iipoprotein （Ox-LDL） in order to find the new possible mechanisms that ABCA1 contributes to atherosclerogencsis. Methods： Human vascular endothelial cell ECV304 were cultured. Ox-LDL （30 ng/ml） was added into culture medium,and ECV304 ceils were examined at 3, 6, 12 and 24 hours. The mRNA of ABCA1, ICAM-1 and protein levels of ABCA1, ICAM-1 and IL-1β were determined by real-time fluorescent quantitative reverse transcription-polymerase chain reaction （RT-PCR）, Western blot, or enzyme-linked immunoadsorbent assay （ELISA） methods. After phosphorothioate antisense oligonucleotides of ABCA1 mixture was added to culture medium at a final concentration of 100 nmol/L, the same determinations were repeated. The ceils cultured without Ox-LDL served as negative control. Results：Comparing to the negative controls, the mRNA and protein quantities of ABCA1, ICAM-1 and IL-1β protein were increased after being incubated with Ox-LDL. After transfection with antisense oligonucleotides of ABCA1, the mRNA expression of ABCA1, ICAM-1 and protein levels of ABCAl,ICAM-l,IL-1β were suppressed. Conclusion： ABCA1 could contribute to atherosclerogenesis probably by increasing the expression of inflammatory cytokines such as IL-1β induced by Ox-LDL in human vascular endothelial cells.