CAJ | 학술논문

目的探讨NRF2基因启动子-617C/A多态性对内毒素(LPS)刺激下酒精性肝病(ALD)患者外周血单个核细胞(PBMC)炎症反应的影响.方法应用Ficoll密度梯度离心法分离82例ALD患者的PBMC,流式细胞仪检测T细胞亚群,同时应用基因测序法检测NRF2基因启动子-617位点的基因多态性,并根据基因测序的结果,将患者分为非突变组(CA和AA)和突变组(CC),以逆转录PCR (RT-PCR)、酶联免疫吸附试验(ELISA)检测LPS刺激下PBMC中NRF2、肿瘤坏死因子(TNF)α、白细胞介素(IL)-1p、1L-10的表达水平.结果 82例患者NRF2-617位点C和A的频率分别为65.9％和34.1％,其中50例(CA:44例；AA:6例)存在突变,32例为非突变(CC).突变与非突变组间比较肝功能、T细胞亚群分布等临床资料差异无统计学意义(P＞0.05).但LPS刺激后,突变组PBMC中NRF2 mRNA表达明显低于非突变组(P＜0.05),而TNFα、IL-1β的mRNA及蛋白表达均明显高于非突变组(P＜0.05),IL-10 mRNA及蛋白表达虽高于后者,但差异无统计学意义(P＞0.05).结论 NRF2基因启动子-617位点C→A突变下调LPS刺激后ALD患者PBMC中NRF2的表达,并加剧促炎反应.
목적 탐토NRF2기인계동자-617C/A다태성대내독소(LPS)자격하주정성간병(ALD)환자외주혈단개핵세포(PBMC)염증반응적영향.방법 응용Ficoll밀도제도리심법분리82례ALD환자적PBMC,류식세포의검측T세포아군,동시응용기인측서법검측NRF2기인계동자-617위점적기인다태성,병근거기인측서적결과,장환자분위비돌변조(CA화AA)화돌변조(CC),이역전록PCR (RT-PCR)、매련면역흡부시험(ELISA)검측LPS자격하PBMC중NRF2、종류배사인자(TNF)α、백세포개소(IL)-1p、1L-10적표체수평.결과 82례환자NRF2-617위점C화A적빈솔분별위65.9％화34.1％,기중50례(CA:44례；AA:6례)존재돌변,32례위비돌변(CC).돌변여비돌변조간비교간공능、T세포아군분포등림상자료차이무통계학의의(P＞0.05).단LPS자격후,돌변조PBMC중NRF2 mRNA표체명현저우비돌변조(P＜0.05),이TNFα、IL-1β적mRNA급단백표체균명현고우비돌변조(P＜0.05),IL-10 mRNA급단백표체수고우후자,단차이무통계학의의(P＞0.05).결론 NRF2기인계동자-617위점C→A돌변하조LPS자격후ALD환자PBMC중NRF2적표체,병가극촉염반응.
Objective To investigate the influence of NRF2 gene polymorphism at locus-617 on inflammatory response of lipopolysaccharide (LPS)-stimulated peripheral blood mononuclear cells (PBMCs) in patients with alcoholic liver disease (ALD).Methods Venous blood samples from 82 patients with ALD were collected and PBMCs were separated using Ficoll density gradient centrifugation.T cell sabgroup was detected by flow cytometry.The polymorphisms in NRF2 gene promoter-617C/A was determined by gene sequencing.According to the results of gene sequencing,patients were divided into non-mutation group (genetype CA and AA) and mutation group (genotype CC).After stimulation with LPS,the expression levels of NRF2,tumor necrosis factor (TNF) α,interleukin (IL)-13 and IL-10 were measured by reverse transcription-PCR (RT-PCR) and enzyme linked immunosorbent assay (ELISA),respectively.Results Among the 82 patients with ALD,32 were homozygous for the C allele (CC),44 heterozygous (CA),and 6 AA.The frequencies of allele C and A were 65.9％ and 34.1％,respectively.There were no differences in clinical data,such as liver fuction and distribution of T cell subsets between the two groups (all P values ＞ 0.05).Under LPS stimulation,the NRF2 mRNA expression in the non-mutation group was significantly higher than that in the mutation group (P ＜ 0.05).The TNFα,IL-1 β mRNA and protein expression in the mutation group were significantly higher than those in the non-mutation group (P ＜ 0.05) and IL-10 mRNA and protein expression of the mutation group was higher than that in the non-mutation group without statistical significance (P ＞0.05).Conclusion The gene promoter NRF2-617C mutated to A in LPS-stimulated PBMC of patients with ALD significantly decreases the expression of NRF2 and releases early proinflammatory cytokines.