高迁移率族蛋白B1对大鼠脾脏树突状细胞细胞因子表达的影响  

Effects of high mobility group box-1 protein on cytokine expression in splenic dendritic cells in rats

作　　者：徐姗[1] 姚咏明[1] 姚凤华[1] 董宁[1] 刘峰[1] 于燕[1] 

机构地区：[1]解放军总医院第一附属医院全军烧伤研究所基础部,北京100037

出　　处：《中华急诊医学杂志》2009年第2期127-131,共5页Chinese Journal of Emergency Medicine

基　　金：国家重点基础研究发展规划项目（2005CB522602）;国家自然科学基金资助项目（30672178,30872683,30800437）

摘　　要：目的观察高迁移率族蛋白B1（HMGB1）对树突状细胞（dendritic cells，DC）细胞因子蛋白合成和基因表达的影响。方法分离正常Wistar大鼠脾脏DC后置于96孔培养板（1×10^5／孔），给予重组HMGB1刺激，研究HMGB1刺激与TNF-α，IL-12蛋白合成和基因表达的时间-效应关系，24孔细胞随机分为6组：正常对照24h组（4孔／组）、正常对照48h组（4孔／组）、正常对照72h组（4孔／组）、HMGB1 24h组（4孔／组）、HMGB1 48h组（4孔／组）和HMGB1 72h组（4孔／组），后3组分别以1μg／mL HMGB1刺激。刺激相应时间检测TNF-α，IL-12 mRNA的表达和蛋白水平。研究HMGB1刺激与TNF-α，IL-12蛋白合成和基因表达的剂量-效应关系，16孔细胞随机分为4组：正常对照组（4孔／组）、0．1μg/mL组（4孔／组）、1μg／mL组（4孔／组）和10μg/mL组（4孔／组），分别以相应剂量HMGB1刺激。刺激后48h后检测TNF-α、IL-12 mRNA的表达和蛋白水平。应用Promega公司mRNA提取试剂盒裂解收集的DC，提取细胞mRNA。采用SYBR Green real-time（实时荧光定量）PCR技术检测TNF-α mRNA，IL-12 mRNA表达水平。以三磷酸甘油脱氢酶（GAPDH）作为内参对照。扩增产物经Fast 7500 real-time PCR仪处理，作相对定量（RQ）分析。以ELISA试剂盒检测各组上清中IL-12，TNF-α蛋白水平。数据进行单因素方差分析，以P〈0．05为差异具有统计学意义。结果1μg／mL HMGB1刺激后，脾脏DC IL-12，TNF-α蛋白合成和基因表达均分别于24～72h明显上调（P〈0．05或P〈0．01），其中以作用48h后其表达上调尤为显著（P〈0．01）；0．1μg／mL，1μg／mL，10μg／mL HMGB1刺激48h均可诱导DC IL-12、TNF-α蛋白合成和基因表达增强（P〈0．01），其中HMGB1浓度在1μg／mL时，DC IL-12和TNF-α蛋白合成和基因表达表达最明显（P〈0．01）。结论HMGB1诱导DC成熟分化过程中能促进DC合成、释放IL-12和TNF-α，从而�Objective To investigate the effect of high mobility group box-1 protein （HMGB1） on cytokine expression in splenic dendritic cell （DCs）. Method DCs isolated from the spleens of male Wistar rats were seeded on 96-well （ 1×10^5 cells/well） cell culture plates＇, and the cells were stimulated with HMGB1 for various length of time or in different concentrations. （ 1 ） The time-dependent response between HMGB1 and tumor necrosis factor-α（TNF-α） as well as interleukin-12 （IL-12） gene/protein expressions： 24 wells of DCs were dividedly into six groups including 24 h-normal controls （ n = 4）, 48 h-normal controls （ n = 4）, 72 h-normal controls （ n = 4）, and 24 h-HMGB1 treated group （ n = 4）, 48 h-HMGB1 treated group （ n = 4） as well as 72 h-HMGB1 treated group （ n = 4）, respectively. Among three HMGB1-treated groups, DCs were stimulated by 1 μg/mL HMGB1.DCs were denatured in cell culture plates to determine gene expression of IL-12 as well as TNF-α, and supematants were harvested to determine IL-12 as well as TNF-α protein levels. （2） The dose-dependent response between HMGB1 and TNF-α as well as IL-12 gene/protein expressions： 16 wells of DCs were dividedly into four groups includingnormal controls （ n = 4）, 0.1 μg/mL HMGB1 treated group （ n = 4）, 1 μg/mL HMGB1 treated group （ n = 4）, and 10μg/mL HMGB1 treated group （ n = 4）, respectively. After stimulated for 48 h, DCs were denatured in cell culture plates to determine gene expression of IL-12 as well as TNF-α, and supematants were harvested to determine IL-12 as well as TNF-α protein levels. Total RNA was extracted from cells using the single-step technique of acid guanidinium thiocyanate-chloroform extraction according to the manufacturer＇s instruction （Promega, Madison, WI）. mRNA for TNF-α and IL-12 were quantified by SYBR Green two-step, real-time reverse transcription-polymerase chain reaction taking glyceraldehyde-3-phosphate dehydrogenase （GAPDH） as an internal standa

关 键 词：高迁移率族蛋白B1 树突状细胞 白细胞介素-12 肿瘤坏死因子-α 免疫调节 

分 类 号：R285.5[医药卫生—中药学]