Construction Of Caveolin-l-shrna Lentivirus And Its Suppression To Hyperpermeability Of Rat Pulmonary Microvascular Endothelial Cell Induced By TNF-α

Objective To construct caveolin-1gene RNA interference lentiviral vector, observingthe influence on pulmonary microvascular permeability while silencing its caveolin-1gene, providing the experiment basis for further research on the mechanism ofALI/ARDS.Metheds Isolating and culturing rat pulmonary microvascular endothelial cells(RPMVECs) in vitro. RT-PCR and Western blot were applied to test the expression ofcaveolin-1in RPMVECs. Caveolin-1gene RNA interference target sequence waschoiced and target DNA was synthesized, double-stranded DNA was formed beforeconnecting to the lentiviral vector pLentilox3.7(pLL3.7) which was digested by Xho Iand Hpa I; the pLL3.7caveolin-1lentiviral vector was transformed into E.coli DH5and was identified by digestion of restrictive endonucleases Xba I and Xho I andsequencing after plasmid was extracted.VSV-G,RSV-REV,pMDL and pLL3.7lentiviralvector were contransfected into293T induced by lipofectamine2000, which producedlentivirus; the supernatant was concentrated and diluted into certain ratio.;293T cell wasinfected by the supernatant and expression of GFP was detected by using fluorescencemicroscope, so that the virus titer was evaluated. RPMVEC was infected by caveolin-1shRNA lentivirus.The efficiency of depressing caveolin-1gene was detected by use ofWestern blot. Transendothelial electrical resistance was measured in order to show thedifferent changes of permeability of RPMVEC monolayer， infected by lentivirus ornot，which was treated by TNF-α. Results1.RPMVECs were isolated and cultured in vitro finely.2.The expression ofcaveolin-1in RPMVECs were detected by use of Western blot.3.Caveolin-1shRNAlentiviral vector pLL3.7-caveolin-1was successfully constructed, which was confirmedby digestion and sequencing.4.After concentration, the titer of packaged lentivirus is2×108TU/ml;5. Western blot showed a significantly decreased expression of caveolin-1in RPMVECs infected by caveolin-1-shRNA lentivirus.The level of caveolin-1decreaseto85%of its original level.6.The decreasing TER across RPMVECs monolayersmediated by TNF-α were improved by the inhibition of the expression of caveolin-1.The difference was significant after infection for30min and was maximum after6hours.There is no significant difference after24hours.（P＜0.05）。Conclusions1.The expression of caveolin-1in RPMVECs was certified by use ofWestern blot.2.The caveolin-1RNAi lentivirus was successfully constructed.3.Theexpression of caveolin-1in RPMVECs was effectively inhibited by caveolin-1shRNAlentivirus.4.The increase of pulmonary microvascular permeability induced by TNF-αwas attenuated by depressing the expression of caveolin-1.