黄芪注射液对脓毒症炎症反应调控作用的研究  

Study of regulatory effect of Astragalus injection on inflammatory response of sepsis

作　　者：徐奇奇 高红梅[2] 窦琳[1] 李寅[1] 路玲[1] 

机构地区：[1]天津中医药大学研究生院,天津300193 [2]天津市第一中心医院重症医学科,天津300192

出　　处：《中国中西医结合急救杂志》2017年第2期180-183,共4页Chinese Journal of Integrated Traditional and Western Medicine in Intensive and Critical Care

基　　金：卫生部国家临床重点专科建设项目（2011-873）;天津市中医药管理局中医、中西医结合科研专项课题（13117）;天津市卫生计生委科技基金（2015KZ019）;天津市卫生行业重点攻关项目（16KG106）

摘　　要：目的观察黄芪注射液对脂磷壁酸（LTA）和脂多糖（LPS）刺激人原代巨噬细胞炎性因子表达的影响，探讨黄芪注射液对革兰阳性（G^＋）菌及革兰阴性（G^-）菌脓毒症炎症反应的作用机制。方法使用Pereoll密度梯度离心法分离人外周血单核细胞，以免疫抗体磁珠提纯单核细胞，经12d在重组人粒一巨噬细胞集落刺激因子（rhGM—CSF）诱导下培养为人巨噬细胞。将培养的人巨噬细胞接种于96孔板（每组3孔）和6孔板中（每组3孔），按随机数字表法分为对照组（加入DMEM培养液，每孔200乩）、LTA1mg／L组、LPS0．1mg／L组以及黄芪注射液低（0．1mg／L）、高（0．2mg／L）剂量组。将培养板置于培养箱中孵育24h后，用酶联免疫吸附试验（ELISA）检测各组上清液中白细胞介素（IL-8、IL-10）的蛋白含量，用实时荧光定量反转录-聚合酶链反应（RT—qPCR）检测IL-8、IL-10的mRNA表达水平。结果LTA、LPS均能明显上调巨噬细胞表达促炎因子IL-8及抗炎因子IL-10水平，培养8h和24h，LTA组、LPS组IL-8和IL-10蛋白以及mRNA表达均较对照组明显升高，以培养24h升高更显著[LTA刺激组：IL-8蛋白（ng／L，×10^3）为41．57±1．90比1．58±0．24，IL-8mRNA（A值）为21．49±8．05比1．00±0．16；IL-10蛋白（ng／L）为5．90±3．02比2．91±1．54，IL-10mRNA（A值）为1．35±0．34比0．95±0．14；LPS刺激组：IL-8蛋白（ng／L，×10^3）为345．00±22．80比5．60±0．31，IL-8mRNA（A值）为29．84±8．93比1．00±0．16，IL-10蛋白（ng／L）为122．37±39．26比44．79±3．67，IL-10mRNA（A值）为7．38±1．58比1．35±0．34，均P〈0．05]；黄芪注射液可使IJTA、LPS刺激巨噬细胞促炎因子IL-8蛋白和mRNA表达降低，抗炎因子IL-10蛋白和mRNA表达升高，以培养24h黄芪注射液高剂量组的变化更为显著[LTA刺激组：IL-8蛋白（ng／L，×10^3）为22．63±1．91比41．57±1．90，IObjective To observe the effect of Astragalus injection on the expressions of inflammatory cytokines in human primary macrophages stimulated by lipoteichoic acid （LTA） and lipopolysaccharide （LPS）, and investigate its effects on inflammatory reactions of Gram-positive （G^＋） and Gram-negative （G^-） bacteria sepsis and its mechanisms. Methods Percoll density gradient centrifugation was used to isolate the human peripheral blood mononuclear cells, then they were purified by immune Anti-Biotin Microbeads with magnetic character and under the induction of recombinant human granulocyte-macrophage colony stimulating factor （rhGM-CSF）, the cells were cultivated for 12 days in vitro, eventually the human monocyte-derived macrophage was formed. The cultured human macrophages were inoculated in 96-well plates （each group 3 wells） and 6-well plates （each group 3 wells）. The cells were divided into control group （200 μL DMEM added in each well）, LTA 1 mg/L group, LPS 0.1 mg/L group and low astragalus injection （0.1 mg/L） and high astragalus injection （0.2 rag/L） dose groups. After the incubator plates were put in an incubator for 24 hours, the protein content of IL-8 and IL-10 in supematant were detected by enzyme-linked immunosorbent assay （ELISA）, and the mRNA expression levels of IL-8 and IL-10 were detected by real time fluorescence quantitative reverse transcription-polymerase chain reaction （RT-qPCR）. Results LTA and LPS all can obviously up-regulate the expression levels of pro-inflammatory factor IL-8 and anti-inflammatory factor IL-10 of macrophage. The expressions of IL-8 and IL-IO protein and mRNA in LTA group and LPS group were significantly higher than those in control group after culture for 8 hours and 24 hours, the degrees of incrementwere more significantly at 24 hours [LTA stimute group： IL-8 protein （ng/L, ×10^3）： 41.57±1.90 vs. 1.58 ±0.24, IL-8 mRNA （A value）： 21.49±8.05 vs. 1.00±0.16; IL-10 protein （ng/L）： 5.90±3.02 vs. 2.91±1.5

关 键 词：脂磷壁酸 脂多糖 人原代巨噬细胞 促炎因子 抗炎因子 

分 类 号：R543.5[医药卫生—心血管疾病]