贵州医科大学学报

2016, v.41;No.186(03) 310-313+329

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布洛芬对MyD88基因沉默中性白细胞中NF-kB蛋白表达的影响
Effect of Ibuprofen on Protein Expression of NF-κB in MyD88-knockdown Human Neutrophils

任飞;王婷婷;王春雷;
REN Fei;WANG Tingting;WANG Chunlei;Department of Geriatrics,the First Hospital of China Medical University;

摘要(Abstract):

目的:研究布洛芬对脂多糖(LPS)介导的My D88基因沉默中性白细胞中一氧化氮(NO)产生能力和TLR4-NF-k B通路中核转录因子kB(NF-kB)蛋白表达的影响。方法:用密度梯度法分离正常健康者外周血中性白细胞并用免疫磁珠纯化,用Nucleofection转染系统将MyD88 siRNA基因转染入纯化后的中性白细胞;用0、5、10、50及100μmol/L布洛芬联合1 mg/L LPS分别刺激正常中性白细胞和已转染的My D88基因沉默的中性白细胞,用流式细胞仪检测已沉默MyD88基因的中性白细胞中NO水平,用Western Blot检测MyD88和NF-k B蛋白表达水平。结果:不同浓度的布洛芬均可以明显抑制正常中性白细胞和MyD88基因沉默中性白细胞产生NO,并呈剂量依赖,50μmol/L布洛芬浓度时抑制率最高;正常人中性白细胞MyD88和NF-κB的表达水平也随布洛芬浓度升高而降低,50μmol/L布洛芬时对MyD88和NF-κB的抑制率最高;选择50μmol/L布洛芬处理正常中性白细胞和MyD88基因沉默的中性白细胞,可明显抑制正常和My D88基因沉默中性白细胞中MyD88-和NF-κB蛋白的表达,与仅加LPS组比较,差异有统计学意义(P<0.01);正常细胞和MyD88基因沉默细胞之间产生NO水平和My D88-、NF-κB蛋白的表达水平比较P>0.05。结论:布洛芬可抑制LPS介导的MyD88基因沉默中性白细胞中NO产生,其机制可能与下调MyD88和NF-κB蛋白水平有关。
Objective: To explore the effect of ibuprofen on NO production in LPS mediated My D88 knockdown human neutrophils and NF-k B protein expression in TLR4-NF-κB pathway. Methods: Ficoll-Hypaque density gradient centrifugation was adopted to isolate the neutrophils from the peripheral blood of healthy people and Easysep Neutrophil Enrichment Kit was used to purify neutrophils. Nucleofection technology was adopted to transfer My D88 siRNA into the purified neutrophils. The cells were inbucated with ibuprofen( 5 μmol / L,10 μmol / L,50 μmol / L and 100 μmol / L) combined with 1mg /L LPS respectively to stimulate normal neutrophils and transfected My D88 gene-silencing neutrophils.Flow cytometry was adopted to detect NO level in My D88 gene-silencing neutrophils and Western Blot was used to detect protein expression level of My D88 and NF-k B. Results: Different concentration of ibuprofen could significantly inhibit NO produced by normal neutrophils and transfected My D88 genesilencing neutrophils in dose-dependent way,with 50 μmol / L of ibuprofen showing the highest inhibition rate. The protein expression level of My D88 and NF-k B in normal neutrophils decreased with the increase of ibuprofen concentration,with 50 μmol / L of ibuprofen showing the highest inhibition rate for protein expression level of My D88 and NF-k B. 50 μmol / L of ibuprofen was selected to stimulate normal neutrophils and transfected My D88 gene-silencing neutrophils and significantly inhibit protein expression level of My D88 and NF-k B. Compared with only LPS group,the differences were statistically significant( P < 0. 01). There was no significant difference in NO level and protein expression level of My D88 and NF-k B between normal neutrophils and transfected My D88 gene-silencing neutrophils( P> 0. 05). Conclusion: Ibuprofen can inhibit LPS-mediated NO produced from My D88 gene-silencing neutrophils and the possible mechanism may be related to down-regulation of protein expression of My D88 and NF-κB.

关键词(KeyWords): 布洛芬;核因子-κB;MyD88;基因沉默;中性白细胞
ibuprofen;nuclear factor-κB;My D88;gene silence;knock-down;neutrophils

Abstract:

Keywords:

基金项目(Foundation): 辽宁省教育厅高校科研基金资助项目(2010686)

作者(Author): 任飞;王婷婷;王春雷;
REN Fei;WANG Tingting;WANG Chunlei;Department of Geriatrics,the First Hospital of China Medical University;

Email:

DOI: 10.19367/j.cnki.1000-2707.2016.03.017

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