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NRF2-617C/A基因多态性对内毒素刺激下酒精性肝病患者外周血单个核细胞炎症反应的影响

Functional polymorphism of NRF2 gene promoter-617C/A in lipopolysaccharide-stimulated peripheral blood mononuclear cellular inflammatory response in patients with alcoholic liver disease

摘要:

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.

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abstracts:

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.

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作者: 赵光举 [1] 陈肖 [1] 李小林 [1] 余毅娟 [1] 洪广亮 [1] 邱俏檬 [1] 李萌芳 [1] 卢中秋 [1]
期刊: 《中华内科杂志》2013年52卷7期 581-584页 MEDLINEISTICPKUCSCD
栏目名称: 论著
DOI: 10.3760/cma.j.issn.0578-1426.2013.07.012
发布时间: 2013-08-06
基金项目:
浙江省科技厅公益性技术应用研究计划项目 浙江省自然科学基金 温州市科技计划项目 温州市高层次人才创新技术重点资助项目
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