中文摘要：

目的 观察肿瘤坏死因子-α（TNF-α）诱导大鼠肺微血管内皮细胞（PMVECs）损伤中膜突蛋白（Moesin）磷酸化水平的变化,探讨Rac1信号通路对Moesin磷酸化的影响.方法 体外培养大鼠PMVECs并传至第3代,分别进行TNF-α时效实验、量效实验以及Rac1信号通路干预实验.① 时效实验：以10μg/L TNF-α分别刺激大鼠PMVECs 0、15、30 min和1、3、6、12 h后,采用蛋白质免疫印迹试验（Western Blot）检测Moesin和磷酸化Moesin（p-Moesin）的蛋白表达.② 量效实验：分别以0、0.1、1、10μg/L TNF-α刺激大鼠PMVECs 6 h后,采用Western Blot检测Moesin和p-Moesin的蛋白表达.③Rac1信号通路干预实验：在PMVECs细胞中分别给予3 mL 200μmol/L的Rac1特异性抑制剂NSC23766预孵0.5 h或200μmol/L的Rac1特异性激动剂O-Me-cAMP预孵1 h,再分别与10μg/L的TNF-α共孵育6 h;同时设立空白对照组及O-Me-cAMP、NSC23766、TNF-α单独处理组;采用Western Blot检测Moesin和p-Moesin的蛋白表达.结果 ① 时效实验结果：以10μg/L TNF-α与大鼠PMVECs共孵育后各时间点Moesin表达量无明显变化;而TNF-α诱导15 min时,p-Moesin表达量即较0 min迅速升高（p-Moesin/Moesin：4.399±0.523比1.000±0.195）,30 min达高峰（6.069±0.557）,1 h后逐渐下降（1、3、6、12 h分别为5.005±0.544、4.599±0.478、1.742±0.288、1.503±0.352）,各时间点间差异有统计学意义（F=15.397,P=0.002）.② 量效实验结果：各剂量TNF-α与大鼠PMVECs共孵育6 h时Moesin表达量无明显变化;而以0.1μg/L TNF-α诱导的p-Moesin表达量即较0μg/L显著升高（p-Moesin/Moesin：2.194±0.430比1.000±0.273）,且随TNF-α剂量增加呈持续升高趋势（1μg/L和10μg/L分别为3.201±0.688、4.413±0.296）,各剂量组间差异有统计学意义（F=92.513,P〈0.001）.③Rac1信号通路干预实验结果：各组间Moesin表达量无明显差异.与空白对照组比较,Rac1特异性激动剂O-Me-cAMP或Rac1特异性抑制剂NSC23766单独刺激均?

英文摘要：

Objective To investigate the role of phosphorylated Moesin （p-Moesin） in the injury of pulmonary microvascular endothelial cells （PMVECs） of rats induced by tumor necrosis factor-α （TNF-α）, and to approach the impact of Rac1 signal pathway on Moesin phosphorylation.Methods PMVECs of rats were culturedin vitroand passed on to the third generation, and the TNF-α time-effect experiment, dose-effect experiment and Rac1 signaling pathway intervention experiment were performed respectively. ① Time-effect experiment： PMVECs were stimulated with 10μg/L TNF-α for 0, 15, 30 minutes and 1, 3, 6, 12 hours, and the protein expressions of Moesin and p-Moesin were determined by Western Blot. ② Dose-effect experiment： PMVECs were stimulated with 0, 0.1, 1, 10μg/L TNF-α for 6 hours, and the protein expressions of Moesin and p-Moesin were determined by Western Blot. ③ Rac1 signaling pathway intervention experiment： PMVECs were divided into two parts, which were pretreated with 3 mL Rac1 specific inhibitor NSC23766 （200μmol/L） for 0.5 hour or Rac1 specific agonist O-Me-cAMP （200μmol/L） for 1 hour, respectively, and then incubated with 10μg/L TNF-α for 6 hours. The PMVECs without treatment were served as blank control group, andthose were treated with only O-Me-cAMP, NSC23766 or TNF-α were served as corresponding groups. The protein expressions of Moesin and p-Moesin were determined by Western Blot.Results ① Time-effect experiment results： the expression of Moesin showed no change among all time points after 10μg/L TNF-α stimulated PMVECs. But the expression of p-Moesin was sharply up-regulated at 15 minutes after TNF-α stimulation as compared with 0 minute （p-Moesin/Moesin： 4.399±0.523 vs. 1.000±0.195）, peaked at 30 minutes （6.069±0.557）, and then gradually decreased after 1 hour （5.005±0.544, 4.599±0.478, 1.742±0.288, 1.503±0.352 at 1, 3, 6, 12 hours, respectively） with significant difference among all time points （F = 15.397,P = 0.002）. ② Dose-effect ex