重组金黄色葡萄球菌杀白细胞毒素对人肺泡巨噬细胞细胞因子表达的影响  

The effect of recombinant panton-valentine leukocidin on the production of cytokines by human alveolar macrophages

作　　者：吴本权[1] 石云锋[1] 黄静[1] 刘慧[1] 张文先[1] 周凤丽[1] 张天托[1] 

机构地区：[1]中山大学附属第三医院呼吸内科,广州510630

出　　处：《中华结核和呼吸杂志》2009年第7期503-507,共5页Chinese Journal of Tuberculosis and Respiratory Diseases

基　　金：基金项目：国家自然科学基金（30670918）;广东省自然科学基金（7001553）

摘　　要：目的应用体外培养人肺泡巨噬细胞（AM），观察重组金黄色葡萄球菌杀白细胞毒素（rPVL）对AM的CD。白细胞介素（IL）-10及肿瘤坏死因子（TNF）-α表达的影响。方法从患者BALF中分离纯化AM，根据作用时间及rPVL浓度不同分为T0N0组[T为时间（h），N为毒素浓度（nmol／L）]、T6N0组、T6N10组、T6N100组、T24N0组、T24N10组、T24N100组共7组。半定量逆转录PCR法测定AM的CD14mRNA水平，双抗夹心ELISA法测定AM培养液上清IL-10及TNF-α浓度。结果rPVL作用后AM的CD14mRNA下降，降低幅度随时间延长和毒素浓度升高而增加。3组空白对照组间差异无统计学意义（F=1．708，P〉0．05）。LN10、T6N100组均较T6N0组低（t=4．132、6．818，均P〈0．001）；T24N10、T24N100组均较T24N0组低（t=7．401、11．415，均P〈0．001），表明毒素作用后CD14表达量降低。T24N10组低于T6N10组、T24N100组低于T6N100组（t=4．692、6．019，均P〈0．001），表明毒素作用后CD14mRNA随时间延长而降低。T6N100组低于T6N10组、T24N100组低于T24N10组（t=2．686、4．014，均P〈0．05），表明CD14mRNA随毒素浓度升高而降低。IL-10浓度T24N10、T24N100组均高于T24N0组（t=4．036、3．941，均P〈0．01），表明IL-10的释放随毒素浓度增加和作用时间延长而增加。TNF-α浓度T24N10组低于T24N0组，而T24N100组高于LNn组（t=2．824、8．468，均P〈0．01），表明低浓度毒素可抑制TNF-α释放而高浓度毒素诱导其大量释放。结论rPVL致AM的CD14表达下降并致AM促炎和抗炎因子表达异常，可致AM功能缺陷引起宿主免疫抑制，不利于炎性细胞对病原菌的清除，可能是杀白细胞毒素金黄色葡萄球菌感染，尤其是重症坏死性肺炎病死率高的重要原因之一。Objective To investigate the effect of recombinant panton-valentine leukocidin （rPVL） on the regulation of human alveolar macrophage CD14 and IL-10 and TNF-α. Methods Human alveolar macrophages（AM） were purified and cultured from bronchoalveolar lavage fluid. Each sample was divided into groups according to different concentrations and exposure times of rPVL. Semi-quantitative RT-PCR was used to evaluate the CO14 mRNA levels and Double-antibody-sandwich-ELISA was used to measure the IL-10 and TNF-α levels in AM cultures. Results CD14 mRNA decreased after rPVL treatment in time-and concentration dependent manners. There were no statistically significant differences in CD14 mRNA among the blank control groups （F = 1. 708, P 〉0. 05）. CD14 mRNA in the T6N10 group and the T6N100 group（ T= time in hours, N = concentration of rPVL/nmol/L） decreased as compared to the T6N0 group （t = 4. 132, 6. 818 ,both P 〈0. 001 ） , and that in the T24N10 group and the T24N100 group also decreased as compared to the T24N0 group （ t = 7. 401,11. 415, both P 〈 0. 001 ） , indicating that the expression of CD14 was downregulated by rPVL treatment. There were also statistically significant differences in CD14 mRNA between T6N10 and T24N100 groups, T6N100 and T24N100 groups （t =4. 692, 6. 019 ,both P 〈0. 001 ） , T6N10 and T6N100 groups, T24 N10 and T24 N100 groups （ t = 2. 686, 4. 014, P 〈 0. 01 respectively）, indicating that the expression of CD14 decreased as the treatment time and the concentration of rPVL increased. The IL-10 concentrations of the T24N10 and T24N100 groups increased as compared to the T24N0 group（t = 4. 036,3. 941 ,both P 〈0. 01 ） in time-deoendent and concentration-dependent manners with rPVL treatment. The TNF-α concentration of the T24N10 group decreased while that of the T24N100 group increased as compared to the T24N0 group （t = 2. 824, 8. 468, both P 〈0. 01, respectively） ,indicating that a lower concentration of rPVL inhibited TNF-α release while a higher c

关 键 词：金黄色葡萄球菌杀白细胞毒素 巨噬细胞 CD14 细胞因子 

分 类 号：R686[医药卫生—骨科学]