论文部分内容阅读
目的探讨核因子κB1基因(NFKB1)启动子区-94位点插入/缺失ATTG碱基(-94ins/del ATTG)多态性在氧化应激中对人原代脐静脉内皮细胞(HUVEC)NF-κB信号通路中P50、P65水平的影响。方法分离并培养HUVEC,采用限制性片段长度多态性聚合酶链反应(PCR-RFLP)法将HUVEC分为插入型(Ⅱ型)、缺失型(DD型)以及杂合子型(ID型),并依据其基因型分型在空白组及H2O2诱导组中分为Ⅱ型、ID型、DD型3个亚组;H2O2建立HUVEC氧化应激模型,采用CCK-8法检测细胞增殖活性选取最适宜的H2O2诱导浓度及时间;Western blot法检测各组HUVEC总蛋白及核蛋白P50、P65蛋白水平。结果共收集23例HUVEC,其中Ⅱ型8例、ID型9例、DD型6例;CCK-8试剂盒确定H2O2诱导浓度为200μmol/L,诱导时间为12 h;在空白组及H2O2诱导组中纯合子DD型P50蛋白水平均较Ⅱ型显著降低。结论 NFKB1(rs28362491)基因多态性对P50蛋白表达影响显著。
Objective To investigate the effect of NF-κB1 gene (-94ins / del ATTG) insertion / deletion at the -94 site on NF-κB1 gene NF-κB1 expression in human umbilical vein endothelial cells (HUVECs) κB signaling pathway P50, P65 levels. Methods HUVECs were isolated and cultured. HUVECs were divided into insert type (Ⅱ), deletion type (DD) and heterozygous (ID) by restriction fragment length polymorphism polymerase chain reaction (PCR-RFLP) According to their genotypes, they were divided into three subgroups: type Ⅱ, type ID and type DD in blank group and H2O2-induced group. HUVEC oxidative stress model was established by H2O2, and the optimal cell proliferation activity was determined by CCK-8 assay H2O2 concentration and time; Western blot was used to detect the total protein and nuclear protein P50, P65 protein levels of HUVEC. Results A total of 23 cases of HUVEC were collected, including 8 cases of type Ⅱ, 9 cases of ID and 6 cases of DD. The concentration of H2O2 induced by CCK-8 was 200μmol / L and the induction time was 12 hours. Homozygous DD type P50 protein levels were significantly lower than type Ⅱ. Conclusion The polymorphism of NFKB1 (rs28362491) has a significant effect on the expression of P50 protein.