TNF-α对肺微血管内皮细胞ERM蛋白表达的研究

Study of the level of ERM proteins in pulmonary microvascular endothelial cells induced by tumor necrosis factor-a

目的观察肿瘤坏死因子-a（TNF-a）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-a量效组（0、0．1、1、10μg／L TNF—a与PMVEC孵育60min）、TNF-a时效组（10μg／LTNF-a分别与PMVEC孵育0、15、30、60、90、120、180min）和ROCK抑制剂（Y-27632）干预组：分别以10μg/L的TNF-a,和30μmol／L Y-27632＋10μg／LTNF-a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-a浓度（0、0．1、1、10μg/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15min开始上升（0．777±0．151），90min达高峰（1．295±0．176），之后渐下降，120min（0．802±0．139），180min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-a．刺激，p-ERM相对表达量（0．634±0．112）较单独TNF-a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-a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。

Objective To investigate the effect of tumor necrosis factor - α （TNF - eL） 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 mierovascular 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 -a） 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 rain 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-a gradually up - regulated the level of p- ERM proteins in a dose -dependent manner [0 μg/LTNF-a group： （0. 648±0. 102）, 0. 1 μg/LTNF-a group： （0.728±0.082） , 1 μg/LTNF-a group： （0.926 ±0. 121） , 10 μg/LTNF-a 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 - a, the level of p - ERM proteins caused a marked attenuation of TNF-a stimulation [ （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-a 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.