中华急诊医学杂志
中華急診醫學雜誌
중화급진의학잡지
CHINESE JOURNAL OF EMERGENCY MEDICINE
2015年
6期
612-616
,共5页
肿瘤坏死因子-α%肺微血管内皮细胞%埃兹蛋白-根蛋白-膜突蛋白%炎症%急性呼吸窘迫综合征%Rho相关激酶
腫瘤壞死因子-α%肺微血管內皮細胞%埃玆蛋白-根蛋白-膜突蛋白%炎癥%急性呼吸窘迫綜閤徵%Rho相關激酶
종류배사인자-α%폐미혈관내피세포%애자단백-근단백-막돌단백%염증%급성호흡군박종합정%Rho상관격매
TNF-α%Pulmonary microvascular endothelial cell%Ezrin-Radixin-Moesin%Inflammation%Acute respiratory distress syndrome%Rho kinase
目的 观察肿瘤坏死因子-α(TNF-α)对大鼠肺微血管内皮细胞(PMVEC)表达埃兹蛋白-根蛋白-膜突蛋白(ezrin-radixin-moesin,ERM)及磷酸化ERM蛋白(p-ERM)的影响,并初步探讨Rho激酶(ROCK)与ERM蛋白磷酸化的关系.方法 体外培养大鼠PMVEC,随机(随机数字法)分为TNF-α量效组(0、0.1、1、10 μg/L TNF-α与PMVEC孵育60 min)、TNF-α时效组(10 μg/LTNF-α分别与PMVEC孵育0、15、30、60、90、120、180 min)和ROCK抑制剂(Y-27632)干预组:分别以10μg/L的TNF-α和30 μmol/L Y-27632+ 10 μg/LTNF-α与PMVEC孵育60min.Western印迹检测ERM蛋白及p-ERM相对表达量.采用SPSS 16.0软件进行分析,多组变量间比较采用单因素方差分析,以P＜ 0.05为差异具有统计学意义.结果 Western印迹检测到大鼠PMVEC均表达ERM蛋白和p-ERM,量效组p-ERM表达量随TNF-α浓度(0、0.1、1、10 μg/L)增加逐渐升高,分别为0.648±0.102、0.728±0.082、0.926±0.121、1.245±0.134(均P=0.000).时效组p-ERM相对表达量于15 min开始上升(0.777±0.151),90 min达高峰(1.295±0.176),之后渐下降,120 min (0.802±0.139),180 min仍维持较高水平(0.769±0.128),分别与未刺激0 min组(0.631±0.123)比较,P=0.004,0.000,0.001,0.016.ROCK抑制剂预处理PMVEC后再给予TNF-α刺激,p-ERM相对表达量(0.634±0.112)较单独TNF-α刺激组(0.875±0.164)显著减少(P =0.002),而较单独ROCK抑制剂组(0.661±0.108)和未处理组(0.654±0.125)差异无统计学意义(分别为P=0.973,P=0.900).结论 TNF-α诱导大鼠PMVEC中的ERM蛋白磷酸化表达增加,ROCK参与其磷酸化调控.
目的 觀察腫瘤壞死因子-α(TNF-α)對大鼠肺微血管內皮細胞(PMVEC)錶達埃玆蛋白-根蛋白-膜突蛋白(ezrin-radixin-moesin,ERM)及燐痠化ERM蛋白(p-ERM)的影響,併初步探討Rho激酶(ROCK)與ERM蛋白燐痠化的關繫.方法 體外培養大鼠PMVEC,隨機(隨機數字法)分為TNF-α量效組(0、0.1、1、10 μg/L TNF-α與PMVEC孵育60 min)、TNF-α時效組(10 μg/LTNF-α分彆與PMVEC孵育0、15、30、60、90、120、180 min)和ROCK抑製劑(Y-27632)榦預組:分彆以10μg/L的TNF-α和30 μmol/L Y-27632+ 10 μg/LTNF-α與PMVEC孵育60min.Western印跡檢測ERM蛋白及p-ERM相對錶達量.採用SPSS 16.0軟件進行分析,多組變量間比較採用單因素方差分析,以P＜ 0.05為差異具有統計學意義.結果 Western印跡檢測到大鼠PMVEC均錶達ERM蛋白和p-ERM,量效組p-ERM錶達量隨TNF-α濃度(0、0.1、1、10 μg/L)增加逐漸升高,分彆為0.648±0.102、0.728±0.082、0.926±0.121、1.245±0.134(均P=0.000).時效組p-ERM相對錶達量于15 min開始上升(0.777±0.151),90 min達高峰(1.295±0.176),之後漸下降,120 min (0.802±0.139),180 min仍維持較高水平(0.769±0.128),分彆與未刺激0 min組(0.631±0.123)比較,P=0.004,0.000,0.001,0.016.ROCK抑製劑預處理PMVEC後再給予TNF-α刺激,p-ERM相對錶達量(0.634±0.112)較單獨TNF-α刺激組(0.875±0.164)顯著減少(P =0.002),而較單獨ROCK抑製劑組(0.661±0.108)和未處理組(0.654±0.125)差異無統計學意義(分彆為P=0.973,P=0.900).結論 TNF-α誘導大鼠PMVEC中的ERM蛋白燐痠化錶達增加,ROCK參與其燐痠化調控.
목적 관찰종류배사인자-α(TNF-α)대대서폐미혈관내피세포(PMVEC)표체애자단백-근단백-막돌단백(ezrin-radixin-moesin,ERM)급린산화ERM단백(p-ERM)적영향,병초보탐토Rho격매(ROCK)여ERM단백린산화적관계.방법 체외배양대서PMVEC,수궤(수궤수자법)분위TNF-α량효조(0、0.1、1、10 μg/L TNF-α여PMVEC부육60 min)、TNF-α시효조(10 μg/LTNF-α분별여PMVEC부육0、15、30、60、90、120、180 min)화ROCK억제제(Y-27632)간예조:분별이10μg/L적TNF-α화30 μmol/L Y-27632+ 10 μg/LTNF-α여PMVEC부육60min.Western인적검측ERM단백급p-ERM상대표체량.채용SPSS 16.0연건진행분석,다조변량간비교채용단인소방차분석,이P＜ 0.05위차이구유통계학의의.결과 Western인적검측도대서PMVEC균표체ERM단백화p-ERM,량효조p-ERM표체량수TNF-α농도(0、0.1、1、10 μg/L)증가축점승고,분별위0.648±0.102、0.728±0.082、0.926±0.121、1.245±0.134(균P=0.000).시효조p-ERM상대표체량우15 min개시상승(0.777±0.151),90 min체고봉(1.295±0.176),지후점하강,120 min (0.802±0.139),180 min잉유지교고수평(0.769±0.128),분별여미자격0 min조(0.631±0.123)비교,P=0.004,0.000,0.001,0.016.ROCK억제제예처리PMVEC후재급여TNF-α자격,p-ERM상대표체량(0.634±0.112)교단독TNF-α자격조(0.875±0.164)현저감소(P =0.002),이교단독ROCK억제제조(0.661±0.108)화미처리조(0.654±0.125)차이무통계학의의(분별위P=0.973,P=0.900).결론 TNF-α유도대서PMVEC중적ERM단백린산화표체증가,ROCK삼여기린산화조공.
Objective To investigate the effect of tumor necrosis factor-α (TNF-α) on the levels of ezrin-radixin-moesin (ERM) proteins and the phosphorylated ERM proteins (p-ERM) in rat pulmonary microvascular endothelial cells (PMVEC),and to explore Rho kinase (ROCK) influencing on modulation of the ERM proteins phosphorylation.Methods Cultured rat pulmonary microvascular endothelial cells were randomly divided into dose-dependent and time-dependent groups.In dose-dependent group,cells were cultured with different doses of TNF-α (0,0.1,1,10 μg/LTNF-α) for 60 min.In time-dependent group,cells were cultured with TNF-α (10 μg/L) for 0,15,30,60,90,120,180 min.In ROCK inhibitor (Y27632) intervention group,cells were cultured with TNF-α (10 μg/L) or Y27632 (30 μmol/L) + TNF-α (10 μg/L) for 60 min respectively.The levels of ERM proteins and p-ERM were determined by western blot.One way analysis of variance (ANOVA) was employed for statistical analysis by using SPSS version 16.0 software to compare values among all groups.A significant difference was presumed as a P value ＜ 0.05.Results Western blot revealed that ERM and p -ERM proteins were present in rat PMVEC.Stimulation withTNF-α gradually up-regulated the level of pERM proteins in a dose-dependent manner [0 μg/LTNF-α group:(0.648 ± 0.102),0.1 μg/LTNF-αgroup:(0.728-±0.082),1 μg/LTNF-α group:(0.926±0.121),10 μg/LTNF-α group:(1.245 ±0.134),all P =0.000].In time-dependent group,the level of p-ERM proteins rose at 15 min (0.777 ±0.151),peaked at 90 min (1.295 ±0.176),then decreased gradually at 120 min (0.802 ±0.139),but stayed higher level at 180 min (0.669 ±0.128) than that in un-stimulated 0 min group (0.631 ±0.123,P=0.004,0.000,0.001,0.016,respectively).When PMVEC pre-incubated with ROCK inhibitor and TNF-t,the level of p-ERM proteins caused a marked attenuation of TNF-αstimulation [(0.634 ± 0.112) vs.(0.875 ± 0.164),P =0.002],however,there are no significant differences compared to ROCK inhibitor alone group (0.661 ± 0.108) and no intervention group (0.654 ± 0.125),P =0.973,P =0.900,respectively).Conclusions TNF-α could induce up-regulation of the level of the phosphorylated ERM proteins in rat PMVEC,and ROCK signal molecules might involve in modulation of the ERM proteins phosphorylation.
