CAJ | 학술논문

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、10 mg/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-4 mRNA的表达；蛋白质免疫印迹法检测核转录因子-κB (NF-κB)抑制蛋白IκB-α和NF-κB p65蛋白水平以及免疫细胞化学染色(NF-κB p65)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10 mg/L的LPS刺激PMVECs,3种细胞因子于2、6和12 h均升高；ICAM-1、TNF-α于2h达分泌高峰,IL-8于12 h达分泌高峰；2 h TLR-4 mRNA表达明显增高达峰值,并持续12 h(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12 h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.
목적 탐토지다당유도적폐미혈관내피세포( PMVECs)염성반응급가능적궤제.방법 분리배양SD대서폐미혈관내피세포,장기분위대조조화LPS (0.01、0.1、1、10 mg/L)간예조.매련면역흡부법검측세포간점부분자-1( ICAM-1),방사면역법검측종류배사인자-α(TNF-α)화백세포개소-8 (IL-8)적수평,실시형광정량PCR검측TLR-4 mRNA적표체；단백질면역인적법검측핵전록인자-κB (NF-κB)억제단백IκB-α화NF-κB p65단백수평이급면역세포화학염색(NF-κB p65)관찰NF-κB적활성변화.결과 여대조조비교,LPS조분비적세포인자현저증가병정제량의뢰성；이10 mg/L적LPS자격PMVECs,3충세포인자우2、6화12 h균승고；ICAM-1、TNF-α우2h체분비고봉,IL-8우12 h체분비고봉；2 h TLR-4 mRNA표체명현증고체봉치,병지속12 h(분별위4.34±1.42、3.62+1.45화3.32±1.36),균고우대조조(1.00±0.00),차이유통계학의의(균P＜0.05)；LPS자격0.5、2、6화12 h후,NF-κB적활성현저증가,표현위억제단백IκB-α신속강해,p65단백동보석출병전입세포핵내.결론 LPS자격PMVECs석방세포인자,기효응병정제량의뢰성,가능시통과격활TLR-4-NF-κB신호통로유도료PMVECs적염성손상.
Objective Lipopolysaccharide (LPS) can activate pulmonary vascular endothelial cells (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 cell 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 performed on isolated PMVECs from SpragueDawley rats. After being identified,PMVECs were divided into 2 groups:a control group,and a LPS (0.01,0.1,1,10 mg/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 immunocytochemical 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 IκB-α and the significant increased release of NF-κB P65 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 subsequently activating NF-κB.