脂多糖致急性肺损伤时Tribbles同源蛋白3表达变化  

Effects of lipopolysaccharide on the expression of tribbles homolog 3 in acute lung injury

作　　者：丁竞帆 尤青海[1] 

机构地区：[1]安徽医科大学第一附属医院呼吸内科,合肥230022

出　　处：《安徽医科大学学报》2017年第8期1142-1147,共6页Acta Universitatis Medicinalis Anhui

基　　金：国家自然科学基金资助项目(编号:81100053)

摘　　要：目的探讨Tribbles同源蛋白3(TRB3)在脂多糖(LPS)致急性肺损伤(ALI)时的变化及其与p38-MAPK信号通路的关系。方法体内实验:复制LPS致ALI大鼠模型,分5 ml/kg生理盐水组和5 ml/kg LPS刺激组,免疫组织化学法检测肺组织中TRB3蛋白表达,RT-PCR检测肺组织TRB3 mRNA表达。体外实验:体外培养大鼠肺微血管内皮细胞(PMVEC),随机分为LPS量效组(0、2、4、10μg/ml LPS分别孵育4 h)、LPS时效组(10μg/ml LPS分别孵育0、4、8、12 h)和p38抑制剂(SB203580)干预组(分为正常对照组、10μg/ml LPS组、10μmol/L SB203580组、10μmol/L SB203580+10μg/ml LPS组)。Western blot法检测TRB3蛋白、p-p38和p38-MAPK表达。结果免疫组织化学法显示大鼠肺泡壁和腺上皮均表达TRB3;RT-PCR法检测大鼠肺组织和大鼠PMVEC均表达TRB3 mRNA;与生理盐水组比较,LPS致ALI大鼠肺组织TRB3 mRNA表达显著增加(t=15.524,P<0.01),LPS刺激的大鼠PMVEC中TRB3 mRNA表达增加(t=7.549,P<0.01);Western blot法显示PMVEC表达TRB3蛋白的表达量随LPS浓度(0、2、4、10μg/ml)增加逐渐升高,差异有统计学意义(F=12.619,P<0.001);时效组TRB3蛋白表达量于4 h达高峰,之后下降,8 h时仍高于0 h,差异有统计学意义(F=11.273,P<0.001)。干预组:与正常对照组比较,10μg/ml LPS组诱导大鼠PMVEC的p-p38、TRB3蛋白表达量增高(t=49.121、15.113,P<0.001);10μmol/L SB203580+10μg/ml LPS组与10μg/ml LPS组比较,p-p38、TRB3蛋白表达量下降(t=7.040、11.900,P<0.05、0.001);10μmol/L SB203580组对大鼠PMVEC表达p-p38及TRB3蛋白无影响,与正常对照组比较,差异无统计学意义。结论 LPS致ALI时TRB3表达增加,TRB3表达受p38-MAPK信号通路调控。Objective To examine the expression of tribbles homologous 3 （TRB3 ） on lipopolysaccharide （ LPS） induced acute lung injury （ALI） and its relationship with p38- mitogen-activated protein kinase （MAPK） pathway. Methods Rats received a intravenous injection by LPS （5 ml/kg） as models of ALI and a intravenous injection by NS （5 ml/kg） as the control. In rat lung tissue the expression of TRB3 protein was examined using immunohisto- chemical staining, the expression of TRB3 mRNA was determined by reverse transcript polymerase chain reaction （RT- PCR） . Cultured rat pulmonary microvascular cells （ PMVEC ） were randomly divided into dose- dependent, time-dependent and intervention groups in vitro. In dose-dependent group, PMVEC were stimulated by various con-centrations of LPS （0,2,4, 10 （ xg/ml） for 4 h , and in time-dependent group PMVEC were challenged by 10 （ xg/ ml LPS for different time （0,4,8,12 h ） . In intervention group, PMVEC grown in normal medium or medium with 10 （ xg/ml LPS for 4 h were pretreated using p38-MAPK inhibitor （10 （ xmol/L SB203580） for 2 h. Western blot was used to examine expression of TRB3 , p- p38 and p38-MAPK. Results Immunohistochemical staining showed that TRB3 protein distributed in rat alveolar walls and glandular epithelium. Increased TRB3 mRNA ex-pression using RT-PCR were found in lung tissue of rats injected by LPS when compared to those in NS group （ t = 15. 524, P 〈0. 01）. Increased TRB3 mRNA expression using RT-PCR had also been found in PMVEC stimulated by LPS when compared to those in NS group （ t =1. 549, P 〈0. 01）. In PMVEC, LPS significantly increased the expression of TRB3 protein in a dose-dependent manner （0, 2 ,4,10 （ xg/ml） after stimulation for 4 h （ F = 12. 619, P 〈 0. 001） . At indicated time-points after PMVEC were challenged by 10 （ jLg/ml LPS, the expression of TRB3 protein raised at 4

关 键 词：脂多糖 血管内皮细胞 Tribbles同源蛋白3 p38-丝裂原激活蛋白激酶 

分 类 号：R345.5[医药卫生—基础医学] R329