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目的观察肺泡巨噬细胞(AM)活化过程和共刺激分子CD40表达变化,探讨其在脂多糖(LPS)诱导的小鼠急性肺损伤(ALI)模型中所发挥的作用。方法BALB/c小鼠分为正常对照组和LPS处理组。光镜观察24、48h肺组织病理变化;RT-PCR测定活化巨噬细胞(activated macrophage,AMφ)中TLR4表达;电泳迁移率变动分析(EMSA)检测AM_φ核提取物中NF-κB的活性;Northern blot检测CD40 mRNA表达,流式细胞术检测CD40蛋白表达;ELISA测定肺泡灌洗液(BALF)中TNF-α、MIP-2及IL-1β的含量。结果小鼠吸入LPS后,肺泡隔断裂、肺间质充血及肺泡腔中性粒细胞浸润,并检测到AMφ表面TLR4的表达、核转录因子NF-κB活性增强、共刺激分子CD40 mRNA和蛋白显著表达,并随着时间延长出现增加趋势,BALF中炎症因子释放增加,正常对照组无明显变化(P<0.05)。结论LPS诱导的ALI中,AM活化和共刺激分子CD40上调,导致瀑链式炎症反应,造成肺急性炎症损伤。
Objective To observe the activation of alveolar macrophages (AM) and the expression of co-stimulatory molecule CD40, and to explore its role in lipopolysaccharide (LPS) induced acute lung injury (ALI) model in mice. Methods BALB / c mice were divided into normal control group and LPS treatment group. The pathological changes of lung tissue were observed with light microscope at 24 and 48 hours. The expression of TLR4 in activated macrophage (AMφ) was detected by RT-PCR. The activity of NF-κB in AM_φ nuclear extract was detected by electrophoretic mobility shift assay (EMSA) The expression of CD40 mRNA was detected by Western blot and the expression of CD40 protein by flow cytometry. The levels of TNF-α, MIP-2 and IL-1β in BALF were measured by ELISA. Results After LPS inhalation, the alveolar septum rupture, pulmonary interstitial congestion and alveolar neutrophil infiltration, and the expression of TLR4 on AMφ surface were observed. The activity of nuclear transcription factor NF-κB was enhanced. The expression of costimulatory molecules CD40 mRNA and protein were significantly increased (P <0.05). The release of inflammatory cytokines increased in BALF with no significant change in normal control group (P <0.05). Conclusions In LPS-induced ALI, CD40 activation is upregulated, leading to cascade inflammation and acute lung injury.