脂多糖诱导肺微血管内皮细胞炎性损伤机制的探讨  

Investigation of inflammatory responses of pulmonary microvascular endothelial cells induced by lipopolysaccharide and mechanism

作　　者：徐淑凤[1] 王平[1] 梁志欣[1] 孙继萍[1] 赵晓巍[1] 李爱民[1] 陈良安[1] 

机构地区：[1]解放军总医院呼吸科,北京100853

出　　处：《中华结核和呼吸杂志》2011年第11期816-820,共5页Chinese Journal of Tuberculosis and Respiratory Diseases

摘　　要：目的探讨脂多糖诱导的肺微血管内皮细胞（PMVECs）炎性反应及可能的机制。方法分离培养SD大鼠肺微血管内皮细胞，将其分为对照组和LPS（0．01、0．1、1、10mg／L）干预组。酶联免疫吸附法检测细胞间黏附分子-1（ICAM-1），放射免疫法检测肿瘤坏死因子-α（TNF—α）和白细胞介素-8（IL-8）的水平，实时荧光定量PCR检测TLR-4mRNA的表达；蛋白质免疫印迹法检测核转录因子-KB（NF-κB）抑制蛋白IKB-α和NF—κBp65蛋白水平以及免疫细胞化学染色（NF—κBp65）观察NF—κB的活性变化。结果与对照组比较，LPS组分泌的细胞因子显著增加并呈剂量依赖性；以10mg／L的LPS刺激PMVECs，3种细胞因子于2、6和12h均升高；ICAM-1、TNF—α于2h达分泌高峰，IL-8于12h达分泌高峰；2hTLR-4mRNA表达明显增高达峰值，并持续12h（分别为4．34±1．42、3．62±1．45和3．32±1．36），均高于对照组（1．00±0．00），差异有统计学意义（均P〈0．05）；LPS刺激0．5、2、6和12h后，NF—κB的活性显著增加，表现为抑制蛋白IκB—α迅速降解，p65蛋白同步释出并转入细胞核内。结论LPS刺激PMVECs释放细胞因子，其效应并呈剂量依赖性，可能是通过激活TLR-4-NF-κB信号通路诱导了PMVECs的炎性损伤。Objective Lipopolysaccharide （LPS） can activate pulmonary vascular endothelial ceils （PMVECs） and induce inflammatory injury. Toll-like receptor-4 （TLR-4） is integrally involved in LPS signaling and has a requisite role in the activation of NF-κB. NF-κB is a key intercellular signaling event that mediates ceil inflammatory responses. The aim of the study was to investigate in an in vitro model the inflammatory responses of PMVECs induced by LPS and the probable mechanism underlying the observed inflammatory responses. Methods The present study was perfolxned on isolated PMVECs from Sprague- Dawley rats. After being identified, PMVECs were divided into 2 groups： a control group, and a LPS （0. 01, 0. 1, 1, 10 rag/L） intervention group. ICAM-1, TNF-α and IL-8 were detected by ELISA or radioimmunological methods. The expression of TLR-4 mRNA was detected by real time PCR. The activation of NF-κB was detected by Western blot （proteins of I-κBα and NF-κB p65 ） and imnmnocytochemical staining （NF-κB p65）. Results Compared with the control group, cytokines secreted from PMVECs- stimulated by LPS were increased in a dose-dependent manner. When stimulated with LPS 10 mg/L for 2, 6 and 12 h, cytokines measured were all increased. ICAM-1 and TNF-α were significantly increased and peaked after 2 h before gradually declining at 6 and 12 h. IL-8 was higher after 2 h, which continued up to 12 h. The expression of TLR-4 mRNA was significantly higher and peaked after 2 h and continued to 12 h （4. 34 ±1.42,3.62 ±1.45,3.32 ±1.36 ）, which were all higher than that of the control group （ 1.00 ±0. 00, P〈0. 05）. Meanwhile, NF-κB was activated at 0.5, 2, 6 and 12 h indicated by the significant degradation of IKB-α and the significant increased release of NF-κB 1）65 and its subsequent translocation into the nucleus with approximately synchronized. Conclusion Taken together, the results demonstrated that LPS was able to induce PMVECs inflammatory injury via activating TLR-4 and subseque

关 键 词：脂多糖类 TOLL样受体4 NF—κB 肺微血管内皮细胞 

分 类 号：R285.5[医药卫生—中药学]