LBP对LPS刺激巨噬细胞分泌细胞因子的调节作用  

Regulation effects of LBP on activating macrophages to secrete cytokines

作　　者：舒旷怡[1] 张颖[2] 杨锦红[1] 任凭[3] 杨爱平[2] 余玲玲[1] 李向阳[1] 

机构地区：[1]温州医学院附属第二医院检验科,温州325027 [2]温州医学院第二临床学院,温州325027 [3]温州医学院检验学院生命科学学院联合学院,温州325027

出　　处：《解放军医学杂志》2010年第8期937-941,共5页Medical Journal of Chinese People's Liberation Army

基　　金：浙江省自然科学基金(Y2080596)

摘　　要：目的采用脂多糖结合蛋白(LBP)和脂多糖(LPS)以不同比例、不同方式刺激THP-1细胞分泌细胞因子,探讨LBP在LPS转运和活化过程中的调节作用,以及单核巨噬细胞受LPS活化后其炎性和负炎性因子分泌的平衡情况。方法将THP-1细胞进行增殖和传代后,经PMA刺激,转化为巨噬细胞。实验分为4个组:LPS组(用不同浓度LPS单独刺激)、LBP组(用不同浓度LBP单独刺激)、LBP/LPS预孵育组(LBP和LPS以不同浓度比例混合,37℃孵育15min)、LBP/LPS不孵育组(先加一定浓度LPS,再以不同浓度比例加入LBP)。分别培养4、12、24h后,吸出上清液,发光法检测TNF-α,IL-10和IL-6水平,并进行统计学分析。结果 THP-1细胞为悬浮生长,用PMA诱导转化后变为巨噬细胞,变为贴壁生长。分别用1、10、100、1000ng/mlLPS单独刺激巨噬细胞分泌TNF-α,1ng/ml和其他组间均有统计学差异(P<0.05),其余3组间无统计学差异(P>0.05)。用LBP刺激巨噬细胞,LBP浓度为1000ng/ml时,TNF-α和IL-6分泌达到最高。LBP/LPS不孵育组中,TNF-α和IL-6分泌曲线在LBP/LPS为10出现最高峰。LBP/LPS预孵育组的TNF-α和IL-6分泌曲线较平缓,无明显分泌高峰。LPS浓度为1000ng/ml时IL-10为8±0pg/ml,其余组IL-10均小于5pg/ml。经析因分析,LPS和LBP之间有交互关系(F=3.425,P<0.01),两者是否预孵育和培养时间有交互作用(F=4.240,P=0.026),LBP浓度和是否预孵育之间有交互作用(F=4.896,P<0.01)。LPS浓度和是否预孵育,LPS浓度和培养时间,LBP浓度和培养时间均无明显交互作用(P>0.05)。结论 LPS是活化单核巨噬细胞的重要物质,LPS在高浓度时刺激巨噬细胞分泌细胞因子具有饱和现象。LBP本身在高浓度时具有刺激巨噬细胞的活性,低浓度的LBP能增强LPS对巨噬细胞的活化,高浓度LBP则起到负性调节作用。体外单核细胞经PMA分化成巨噬细胞后,再用LPS刺激,IL-10无明显分泌,引起炎性和负炎性因子的严重失衡。Objective To study the regulatory effects of lipopolysaccharide （LPS） binding protein （LBP） on the process of LPS transference and activation effects,and observe the secretion balance of macrophage cells in secreting pro-inflammatory cytokines and anti-inflammatory cytokines as a result of stimulation by LPS. Methods After proliferation and passage,the human THP-1 cells were induced into macrophages by PMA. Experiment was divided into four groups：LPS group,LBP group,LBP/LPS pre-incubation group,and LBP/LPS without pre-incubation group. Cells were cultured for 4,12 and 24 hours,respectively,and the supernatants were then removed. TNF-α,IL-10 and IL-6 contents were determined by luminescence assay,the results were then statistically analyzed. Results a） THP-1 cells grew as suspended cells,and transferred as adherent macrophages after PMA inducement. b） LPS in respective concentration of 1ng/ml,10ng/ml,100ng/ml and 1000ng/ml was used alone in stimulating macrophages,the secretion levels of TNF-α differed significantly between 1ng/ml group and the other 3 groups （P0.05）,while no significant difference was found among the other 3 groups （P0.05）. c） TNF-α and IL-6 secretion reached a maximum value when macrophages were stimulated by LBP in concentration of 1000ng/ml. d） In LBP/LPS without pre-incubation group,the peak value of TNF-α and IL-6 secretion was fourd when the LBP/LPS was equal to 10; the TNF-α and IL-6 secretion curve was more or less flat without a peak in LBP/LPS pre-incubation group; IL-10 was less than 5pg/ml. e） Factorial analysis revealed that no interaction existed between LPS and LBP （F=3.425,P0.01）,incubation and stimulation time （F=4.240,P=0.026）,as well as between LBP and incubation time （F=4.896,P0.01）. Conclusions LPS plays an important role in activating macrophages. LBP,which may stimulate macrophages in a high concentration,shows positive regulatory effect in low dose,while high dose of LPS shows a negative regulatory effert. After PMA differentiat

关 键 词：脂多糖类 脂多糖结合蛋白 肿瘤坏死因子α 白细胞介素-10 白细胞介素-6 

分 类 号：R349.5[医药卫生—基础医学]