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炎症细胞诱导的活性氧类生成和肠道氧化应激与慢性炎症性肠疾病以及结直肠肿瘤的发病密切相关.目前,核因子κB(NF-κB)信号通路在氧化应激反应、结直肠炎症和肿瘤发生中的作用还未研究清楚.本研究将化学合成的一对编码小干扰RNA序列、靶向人NF-κB基因长60 bp寡核苷酸链定向克隆至pSUPER小干扰RNA表达载体中,经单酶切、双酶切及测序证实重组RNA干扰载体构建成功.将构建成功的质粒转染至结肠上皮细胞HCT116中敲减p65,分别采用免疫印迹方法检测NF-κB p65蛋白表达水平,(3-(4,5-二甲基噻唑-2)-3,5-二苯基四氮唑溴盐(MTT)方法检测细胞存活情况.结果显示,pSUPER-NF-κB p65载体可特异性下调NF-κB p65蛋白表达;下调p65表达可导致过氧化氢诱导的HCT116内活性氧类物质生成增高,存活细胞数目显著减少,氧化损伤加重.研究表明,在人结肠上皮细胞内NF-κB p65通路的抑制显著加重了结肠上皮细胞氧化损伤情况.
Inflammatory cells induce reactive oxygen species production and intestinal oxidative stress and chronic inflammatory bowel disease and the incidence of colorectal cancer are closely related. At present, nuclear factor kappa B (NF-κB) signaling pathway in oxidative stress, colorectal inflammation And tumorigenesis has not been studied.In this study, a pair of chemically synthesized small interfering RNA sequences and a 60 bp oligonucleotide targeting human NF-κB gene were cloned into pSUPER small interfering RNA expression vector , Double digestion and sequencing confirmed that the recombinant RNA interference vector was successfully constructed.Construction of successful plasmid transfected into human colon epithelial cells HCT116 knockdown p65, were detected by Western blot NF-κB p65 protein expression levels, The cell viability was detected by MTT assay. The results showed that the pSUPER-NF-κB p65 vector was specific Down-regulated the expression of p65 protein, down-regulated the expression of p65, which led to the increase of reactive oxygen species in HCT116 induced by hydrogen peroxide, the significant decrease of the number of viable cells and the increase of oxidative damage.Studies showed that NF-κB p65 Inhibition of the pathway was significantly aggravated Oxidative damage of intestinal epithelial cells.