输血相关急性肺损伤对SD大鼠巨噬细胞活化和共刺激分子CD40表达的影响

Expression of co-stimulated molecule CD40 of macrophage on transfusion-related acute lung injury in Sprague Dawley rats

目的观察输血相关急性肺损伤（transfusion—related acute lung injury，TRALI）SD大鼠肺泡巨噬细胞活化及共刺激分子CD40表达，探讨其在TRALI发病机制中的作用。方法SD大鼠60只，随机（随机数字法）分为正常对照组、脂多糖对照组、TRALI组和阳性对照组各15只。正常对照组采用假手术处理；脂多糖对照组大鼠经腹腔注射脂多糖（2ms／kg，≤1min完毕），2h后静脉输注生理盐水（约1mL／只）；TRALI组腹腔注射脂多糖（2mg／kg）2h后静脉输注血浆（约1mL／只）；阳性对照组静脉输注脂多糖（5ms／kg）诱导急性肺损伤。光镜观察肺组织病理变化；应用实时荧光定量聚合酶链反应（RT-PCR）测定活化巨噬细胞（activatedmacrophage，AMφ）中TLR4表达；电泳迁移率变动分析（EMSA）检测AMφ核提取物中NF—KB的活性；Northernblot检测CIM0mRNA表达，流式细胞术检测CD40蛋白表达；酶联免疫吸附试验（ELISA）测定支气管肺泡灌洗液（BALF）中TNF一α、MIP-2及IL-1β的含量。结果TRALI组和阳性对照组大鼠肺泡隔断裂、肺间质充血及肺泡腔中性粒细胞浸润；活化AM表面TLR4的表达升高、NF．KB活性增强、共刺激分子CD40mRNA和蛋白显著表达，与正常对照组和脂多糖对照组比较差异具有统计学意义（均P〈0．01）。TRALI组BALF中TNF-α、MIP-2、IL-1β的表达水平均显著高于正常对照组和脂多糖对照组（P均〈0．05）。结论AMφ能上调其表面共刺激分子的表达，促进炎细胞因子的释放，增强获得性免疫反应介导的急性肺损伤，提示AM活化可能在TRALI的发生过程中发挥一定作用。

Objective To study the activation of alveolar macrophage 13 （AM） and the expression of co-stimulatory molecule CD40 in transfusion-related acute lung injury （TRALI） model in order to illustrate the pathogenesis of TRALI. Methods Sixty SD rats were randomly （random number） divided into normal control group （ n = 15 ） with sham operation using normal saline instead of LPS and plasma, positive control group （n = 15 ） with ALI induced by intravenous infusion of 5 mg/kg lipopolysaccharide （LPS） in equivalent volume of whole blood drawn out）, and TRALI group （n = 15 ） treated by intra-peritoneal injection of 2 mg/ kg LPS 2 h before the transfusion of human plasma （ 1 mL whole blood about 10% of total blood volume drawn out and replaced with 1 mL plasma）, LPS control group （ n = 15 ） treated by intra-peritoneal injection of 2 mg/kg LPS 2 h before saline infusion in equivalent volume of blood drawn out. The pathologic changes of rat lung tissue were observed by HE staining. The expression of TLR4 was examined by RT-PCR. The activation of NF-xB in AM was measured by electrophoresis mobility shift assay （EMSA）. The expression ofCD40 mRNA and CD40 molecule were analyzed by Northern blot and flow cytometry respectively. ELISA was performed to detect the concentration of TNF-α, MIP-2 and IL-1β in broncho-alveolar lavage fluid （BALF）. Results Broken alveolar septa, hyperemia, and massive infiltration of inflammatory cells including the neutrophils were observed in lung tissues of TRALI group. The expression of TLR4 gene was detected in activated macrophage phi （AMφ） of TRALI group rats. The activation of NF-xB was increased in TRALI group rats. The expression of CD40 in AMφ was higher in rats of TRALI group than that in rats of control group and LPS control group. The concentration of TNF-α, MIP-2 and IL-1β were enhanced significantly in BALF of TRALI group rats. Conclusion The activation of AM and up-regulation of costimulatory molecule CD40 induced release of some inflammatory cytokines. It suggested that AM activation may play an important role in the pathogenesis of TRALI.