华支睾吸虫排泄-分泌产物对小鼠巨噬细胞一氧化氮产生和核转录因子κB活化的影响  

Effect of Excretory-secretory Products of Clonorchis sinensis on Nitric Oxide Production and NF-κB Activation in RAW264.7 Mouse Macrophages

作　　者：杨庆利[1,2] 蒋智华[2] 申继清[3] 陈颖丹[1] 周晓农[1] 

机构地区：[1]中国疾病预防控制中心寄生虫病预防控制所卫生部寄生虫病原与媒介生物学重点实验室世界卫生组织疟疾、血吸虫病和丝虫病合作中心,上海200025 [2]广西壮族自治区疾病预防控制中心广西病毒性肝炎防制研究重点实验室,南宁530028 [3]广西医科大学寄生虫学教研室,南宁530021

出　　处：《中国寄生虫学与寄生虫病杂志》2015年第2期96-100,共5页Chinese Journal of Parasitology and Parasitic Diseases

基　　金：国家自然科学基金(No.31260221)~~

摘　　要：目的研究华支睾吸虫(Clonorchis sinensis)排泄-分泌产物(ESPs)对小鼠巨噬细胞系RAW264.7一氧化氮(NO)产生与核转录因子κB(NF-κB)活化的影响。方法用20μg/ml华支睾吸虫ESPs水溶性浓缩物及其有机溶剂提取物(ESPs-ex)和0.1μg/ml明尼苏达沙门氏杆菌脂多糖(LPS-SM)分别刺激RAW264.7细胞,未刺激对照组加入等量Hank’s平衡盐缓冲液(HBSS)。实验同时用0.3 mmol/L诱导型一氧化氮合成酶(i NOS)抑制剂SMT作为干预。细胞培养18 h后,用Griess法检测各组细胞培养上清液NO2-浓度。将p Ni Fty2-SEAP质粒转染至4组刺激后RAW264.7细胞,培养18 h后,检测培养上清液中可溶性胚胎碱性磷酸酶(SEAP)的吸光度(A620值),倒置显微镜观察细胞内SEAP催化显色情况。结果经ESPs-ex和LPS-SM刺激后,细胞培养上清液中NO2-浓度分别为(14.30±1.62)和(14.10±2.17)μmol/L,均较未刺激对照组[(7.70±0.95)μmol/L]显著增加(P<0.05);加入SMT后,两组NO2-浓度显著下降,分别为(8.97±0.81)和(4.96±1.36)μmol/L(均P<0.05)。经ESPs刺激后,上清液中NO2-浓度为(4.06±0.62)μmol/L,较未刺激对照组显著降低(P<0.05);加入SMT后,NO2-浓度为(3.99±0.87)μmol/L,无明显变化(P>0.05)。ESPs刺激后,上清液SEAP的A620值为0.836±0.005,显著高于未刺激对照组(0.097±0.009)(P<0.05);镜下可见细胞内出现广泛、强烈的蓝色显色反应。ESPs-ex和LPS刺激后,上清液SEAP的A620值分别为0.112±0.004和0.116±0.009,略高于未刺激对照组(P>0.05);镜下见部分细胞内出现蓝色显色反应。结论华支睾吸虫ESPs能够促进RAW264.7细胞活化NF-κB,其水溶性浓缩物可抑制NO产生,ESPs-ex和LPS-SM可促进NO产生。Objective To study the role of excretory-secretory products（ESPs） from Clonorchis sinensis in the production of nitric oxide （NO） and the activation of nuclear transcription factor kappa B（NF-κB） in the macrophages of RAW264.7 mouse. Methods 20 μg/ml of C. sinensis EPSs， the organic solvent extracts of EPSs（ESP-ex）， and 0.1 μg/ml of lipopolysaccharide from Salmonella minnesota（LPS-SM） were used as stimulators in co-culture with RAW264.7 mouse macrophages as experimental groups. The Hank’s balanced salt solution（HBSS） served as control. At the same time the RAW264.7 macrophages were stimulated with EPSs， ESP-ex， and LPS-SM， and then added 0.3 mmol/L of SMT， a specific inhibitor of iNOS as the interference groups. After co-culture for 18 days， the concentrations of NO2- in the culture supernatants were detected with Griess regents， and the activation of NF-κB was determined by transfection with a NF-κB-inducible reporter plasmid， pNiFty2-SEAP. The activities of secreted embryonic alkaline phosphatase（SEAP） in culture supernatants were quantified by using HEK-BlueTM detection medium and expressed as the value of optical density at 620 nm（A620 value）. The intercellular activities of SEAP were determined by microscopic observation. Results After stimulation with both ESPs-ex and LPS-SM， the concentrations of NO2- in culture supernatants were（14.30±1.62） and （14.10±2.17） μmol/L， respectively， which were significantly higher than that of the control［（7.70±0.95） μmol/L］（P〈0.05）， and significantly decreased to （8.97±0.81） and （4.96±1.36） μmol/L after adding SMT， respectively（P〈0.05）. However， the concentration of NO2- in ESPs stimulation group ［（4.06±0.62） μmol/L］ was lower than that of the control（P〈0.05）， and almost unchanged［（3.99±0.87） μmol/L］ after adding SMT（P〉0.05）. SEAP activity in ESP group（0.836±0.005） was significantly higher than that of the control ［（0.097

关 键 词：华支睾吸虫 排泄-分泌产物 病原相关分子模式 一氧化氮 核转录因子&kappa B 

分 类 号：R383.22[医药卫生—医学寄生虫学]