Ezrin/Radixin/Moesin 蛋白对肺微血管内皮细胞通透性的调控研究

The key of pathogenesis on acute respiratory distress syndrome(ARDS) is the increase of pulmonary microvascular endothelial cells(PMVEC) permeability,which is related to the cytoskeletal protein and its signal transduction. The aim of this study is firstly to examine the expression, distribution of ezrin, radixin, moesin (ERM) on rat PMVEC, then, investigate the relation of ERM and F-actin in cells. Secondly, the changes of ERM are assessed on the regulation of endothelial hyperpermeability after exposure of PMVEC to lipopolysaccharide (LPS) and tumor necrosis factor-α (TNF-α). The technology of RNA interference through lentiviral vector inhibiting ERM expression and plasmid transfection overexpressing ERM is used to investigate the regulation of those proteins in endothelial permeability, respectively. We also explore influence of protein kinases (Rho-associated kinase, protein kinase C and mitogen activated protein kinases) on ERM. The feedback regulation mechanism of ERM on protein kinases is investigated by RNA interference and plasmid transfection overexpression, respectively. To alleviate the injury of PMVEC induced by inflammation, we intend to intervene in the signal transduction of PMVEC according to different regulatory mechanism of ERM.

肺微血内皮细胞（PMVEC）通透性增加是ARDS发生的关键环节，其发生机制与PMVEC骨架蛋白及相关的信号转导变化密切相关。本课题检测大鼠PMVEC中埃兹蛋白、根蛋白、膜突蛋白（Ezrin, radixin, moesin, ERM）的表达、分布及其与肌动蛋白的关系。观察脂多糖（LPS）、肿瘤坏死因子（TNF-α）等诱导大鼠PMVEC单层通透性增加过程中ERM 3种蛋白的变化。构建ERM 3种蛋白基因RNA干扰慢病毒，观察抑制ERM基因表达后对PMVEC通透性的影响。检测质粒转染高表达ERM蛋白后上述过程的变化。研究Rho相关激酶、PKC、MAPK等对ERM 蛋白的调节作用。分别探讨抑制或高表达ERM 蛋白后对上述蛋白激酶的反馈调节机制，针对ERM蛋白作用环节，采取干预措施影响PMVEC内信号转导，以期减轻炎性介质诱导的PMVEC单层通透性增加。

急性肺损伤过程中，构成交换屏障最内层的肺微血管内皮细胞是受多种炎症因子攻击的效应细胞。本研究通过Western-blot、pull down、RNA干扰、TER等手段对ERM（ezrin/radixin/moesin）蛋白在原代培养的大鼠肺微血管内皮细胞（RPMVEC）炎症微环境下的通透性、细胞骨架、骨架相关蛋白以及其中的信号通路改变中的作用进行研究 ，发现：（1）RPMVEC少量表达ERM及Ezrin、Radixin、Moesin蛋白。（2）LPS刺激RPMVEC不改变ERM及Ezrin、Radixin、Moesin蛋白的总表达量，可呈时间及浓度依赖性改变p-ERM、p-Ezrin、p-Radixin、p-Moesin表达量。10mg/L LPS时间依赖性的增加p-ERM、p-Ezrin、p-Radixin、p-Moesin表达量，30min达高峰，10mg/L的LPS刺激30min，P-E RM，P-Ezrin，P-Radixin，P-Moesin的表达水平高于低浓度组。（3）TNF-α刺激RPMVEC不改变ERM、Ezrin、Moesin蛋白的总表达量，可呈时间及浓度依赖性增加p-ERM、p-Ezrin、p-Moesin的表达量。（4）RhoA、Rac1参与LPS诱导的RPMVEC损伤过程，RhoA抑制剂干预可降低p-ERM表达，激活及抑制Rac1可分别降低和增加p-ERM、p-Moesin、p-Ezrin的表达。（5）在ROCK、Rac1参与TNF-α诱导的RPMVEC损伤模型中，ROCK抑制剂干预可降低p-ERM的表达，激活和抑制Rac1可分别降低和增加p-Moesin、p-Ezrin的表达。（6）抑制Moesin蛋白表达可增加Rac1的活性，从而降低LPS引起RPMVECs单层高通透性。特异性下调Rac1的活性，可削弱抑制Moesin基因表达带来的内皮屏障保护效应。