烧伤大鼠离体肺泡巨噬细胞CD14的表达及分泌IL-6的调控研究

Researches on Effects and Modulation of AM CD14 Expression and IL-6 Level in Burned Rats

目的探讨严重烧伤对大鼠肺泡巨噬细胞(AM)CD14膜蛋白(CD14)和mRNA基因表达变化的影响,以及抗CD14抗体对AM产生白细胞介素-6(IL-6)的调控作用。方法20%Ⅲ°烧伤大鼠检测早期外周血内毒素(LPS)浓度。体外分离培养AM,分成烧伤血清组、血清抗体组、内毒素组、内毒素抗体组。RT-PCR方法观察膜表面CD14 mRNA表达、免疫组织化学方法观察蛋白含量及ELISA方法观察分泌IL-6的变化。烧伤血清刺激体外培养的正常大鼠AM,观察培养上清中IL-6浓度变化及CD14抗体的抑制作用。结果烧伤后外周血LPS各时相点LPS浓度均明显高于对照组,与此相对应,烧伤组大鼠AM各时相点CD14 mRNA表达、蛋白含量均明显增高,AM培养上清中IL-6浓度亦显著增高(P<0.01)。以烧伤血清与AM培养1 h后,烧伤组培养上清中IL-6浓度增加值明显高于对照组(P<0.01),而在CD14抗体存在时,IL-6浓度增加值明显小于烧伤组(P<0.01)。结论严重烧伤后外周血LPS浓度增加,AM膜表面CD14受体亦显著增加,使LPS对免疫系统的激活作用显著增大,AM分泌IL-6明显增加。提示严重烧伤后可以通过调节CD14的作用而减少炎性介质的合成和分泌。

Objective To observe the role of pulmonary alveolar macrophage （AM） CD14 protein in the production of interleukin- 6 （IL-6） after severely burned rats at early stage. Methods SD rats were burned up to 20% of the surfaces with TBSA III degree injury and the dynamic changes in plasma level of LPS werre observed. After the AMs of the burned rats were isolated and cultured, the level of CD14 mRNA expression and the protein produced by AMs were detected with RT-PCR, immunohistochemical and ELISA method, respectively. In vitro, the level of IL-6 in the supernatant of normal rat AMs cultured with post-bum serum with or without CD14 antibody were also detected. Results The level of plasma LPS increased significantly after being severely burned. Correspondingly, the expression of CD14 mRNA, the protein of AMs and level of IL-6 in the supernatant of AMs also increased. When AMs were cultured with post-burn serum, the level of IL-6 in the supernatant increased significantly, which could be reversed at the present of CD14 antibodies. Conclusion After severe burn, the role of LPS stimulation to immune system augments because of the increase of plasma LPS level and AMs CD14 receptor numbers resulting in the increase Of IL-6 secretion. It is indicated that the production of inflammatory cytokines may be inhibited via modulating CD14 signal transduction.