| ObjectiveThe hyperpermeability of pulmonary microvascular endothelia cells,which was one of the most important structures that composed the blood-air barrier, was the pathophysiologic basis of acute respiratory distress syndrome.Inflammatory cytokines induced endothelia cell edema on one hand,which thickened the blood-air barrier that causing low efficiency of diffusion,and activation of inflammatory cells on the other hand,which produced lots of inflammatory mediators that damaged microvascular endothelia cells directly and led to cascade amplification of inflammatory reaction. Furthermore,plenty of inflammatory mediators could induce apoptosis,cytoskeleton rearrangement and altering the interendothelial junctions, which caused hyperpermeability of RPMVECs.Caveolin-1,the structural protein of caveolae,whose scaffolding domain combined with varied signaling molecules,confered the center of multiple signaling pathway to caveolae. Ezrin, radixin and moesin(ERM) were three highly homologous protein members of the FERM superfamily. Fundamentally, they were essential for linking the actin cytoskeleton to the cell membrane and were key organisers of specialized membrane domains. Their function as cytoskeletal linkers placed them at the centre of an elaborate regulatory network of many cellular processes, such as migration,growth and adhesion. The C-terminal domain of ERM proteins could bind to the FERM domain of the same molecule, through selfassociation, to form a monomer or, with another ERM molecule, to form a homo- or heterodimer. This intramolecular interaction led to aclosed conformation or head–tail interaction, and the consequent masking of both the membrane and actin binding sites, resulting in inactivation of the proteins.The study observed the effect of down-regulation of caveolin-1 expression on hyperpermeability of RPMVECs monolayer, rearrangement of cytoskeleton and phosphorylation of ERM induced by TNF-α. The expression of moesin was also depressed in order to study its function in the RPMVECs injury induced by TNF-α.Methods1. primary RPMVECs were isolated and cultured in vitro.2. Different monolayers were established on transwell by use of low expression of caveolin-1 cells or normal expression cells.The monolayer would be given different stimulations according to the study project. Then detected the change of TER and the flux of FITC-BSA.3. The rearrangement of cytoskeleton and change of phosphorylation of ERM in RPMVECs,which were stimulated with different drugs,were observed by laser confocal microscope.4. Detecting the phosphorylation of ERM by western blotting.5. The expression plasmid and lentivirus targeting moesin were constructed. RPMVECs were infected with the lentivirus in order to down regulation of moesin expression.6. Observing the changes of TER in different monolayers composed by low expression of moesin cells or normal expression cells.Results1. RPMVECs were successfully isolated and cultured in vitro and were identified.2. Primary RPMVECs were infected with caveolin-1-sh RNA lentivirus andcontrol-sh RNA lentivirus. After 48 h the cells expressed GFP which was a kind of green fluorescence protein that could be seen by fluorescence microscope. The green fluorescence was brighter at 72 h. Western blot was used to detect the expression of caveolin-1,which began to reduce at 48 h and to 21±6% at 72 h. The expression of caveolin-1 in primary RPMVECs which were infected with control lentivirus had no changes.3. Down-regulating the expression of caveolin-1 led to an mildly increase in mean baseline TER of RPMVECs monolayer compared with control-sh RNA RPMVECs monolayer. All TERs of monolayers which were given stimulations began to decrease at 15 min and dropped to the least at 3 hours compared with control group. Down-regulation of caveolin-1 could attenuate the TER decrease of monolayers which were exposed to TNF-α or PMA. On this basis,low expression of caveolin-1 monolayer,pretreated with BIM for 2 hours could largely reduce the drop of TER induced by TNF-α compared with control monolayer.Over all,PKC inhibitor BIM could attenuate the decline of TER induced by TNF-α and on this basis down-regulation of caveolin-1 could further alleviate it but still lower than the negative control group. The flux of FITC-BSA obviously increased(~8-fold increase) after 4 hours of TNF-α or PMA stimulation compared with control-sh RNA groups. Pre-incubation of monolayer with BIM for 2 hours,the flux of FITC-BSA significantly decreased but still higher than the control-sh RNA group. Down-regulation of caveolin-1 expression could dramatically decrease the flux of FITC-BSA but did not yet decrease to the level of caveolin-1-sh RNA group。4. No stress fiber and low phosphorylation of ERM in the normal condition. F-actin distributed along the membrane in the peripheral cell. Down-regulation of caveolin-1 has few effect on the rearrangement of cytoskeleton and phosphorylation of ERM in the normal condition.5. Down-regulation of caveolin-1 attenuated the formation of fiber stress andphosphorylation of ERM induced by TNF-α(100ng/ml) compared with normal and control-sh RNA cells.6. PKC activator PMA(100nmol/L) induced formation of stress fiber and phosphorylation of ERM more apparently in control-sh RNA cells than in caveolin-1-sh RNA cells. PKC inhibitor BIM(1μmol/L) suppressed the phosphorylation of ERM induced by TNF-α but could not restrain the formation of stress fiber completely in control-sh RNA cells.7. Successfully constructed three recombinant p LL3.7-moesin plasmid and concentrated the packaging lentivirus. The titres were 109TU/ml、109TU/ml、3×109 TU/ml and the down-regulation efficiency were 为 61.5±11.2%、53.6±8.5%、76.1±3.8%.8. Few expression of phosph-ERM in normal RPMVECs. The control RPMVECs exposed to TNF-α for 2 hours,the phosphorylation of ERM increased dramatically compared with negative group. TNF-α induced phosphorylation of ERM in RPMVECs was partly blocked by BIM. Meanwhile BIM could not suppress the phosphorylation of ERM in low expression of caveolin-1 cells exposed to TNF-α.9. Depressing the expression of moesin could alleviate the hyperpermeability of RPMVECs monolayer induced by TNF-α.Conclusion1. Caveolin-1 could attenuate the hyperpermeability of RPMVECs monolayer induced by TNF-α via regulating the activation of PKC.2. The rearrangement of cytoskeleton and phosphorylation of ERM in RPMVECs depended on the existence of caveolin-1.3. Successfully constructed the model of RPMVECs which low expressed moesin.4. Depressing the expression of moesin could alleviate the hyperpermeability ofRPMVECs monolayer induced by TNF-α... |
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