Preliminary Study The Role Of Caveolin-1in Rat Pulmonary Microvascular Endothelial Cell In The Inlfammatory Injury

Objective To investigate the effects of lipopolysaccharide (LPS) on mRNAexpression of caveolin-1(Cav-1) in rat pulmonary microvascular endothelial cell(RPMVEC); and to observe the intervention of protein kinase C（PKC）inhibitor in theCav-1mRNA expression induced by LPS; to further delineate the function andimportance of Cav-1in the pathogenesis of acute lung injury/acute respiratory distresssyndrome（ALI/ARDS）.Methods Isolating and culturing RPMVEC in vitro; Using reverse transcriptpolymerase chain reaction （RT-PCR） and immunization fluorescence methodsrespectively to detect the Cav-1mRNA and related protein expression; Dividing theRPMVEC into concentration-varying group and time-varying group: inconcentration-varying group, culturing RPMVEC with LPS with followingconcentrations,0.1μg/ml,1μg/ml,10μg/ml for6h, in time-varying group, culturingRPMVEC with10μg/ml LPS for1h,2h,3h,6h respectively. Both groups werecompared to the control group. Culturing RPMVEC with PKC inhibitorbis-indolylmaleimide (BIM) together for1h first, then it was cultured with LPS(10μg/ml) for another6h, which was also compared to the control group. In situhybridization(ISH) and JD801morphology imaging analysis system were used to detectthe change of Cav-1mRNA expression in RPMVEC under various conditions.Results1. RPMVEC was successfully isolated and cultured in vitro, which wasidentified by morphology and fluorescein isothiocyanate phytohemagglutinin （FITC-PHA）integration experiment.2. RT-PCR and immunization fluorescencedetected appearance of Cav-1mRNA translation and transcription in RPMVEC.3. Theexpression of Cav-1mRNA in RPMVEC in6h was enhanced distinctively withincreasing LPS concentration from0.1μg/ml to1μg/ml and to10μg/ml, compared withthe results of control group（P <0.05）; The stimulation effects of10μg/ml LPS to theCav-1mRNA expression in RPMVEC increased greatly with time from1h to2h, afterwhich the effects decreased along time to6h. It was worthy to note that at6h theexpression effects was still stronger than the control group（P <0.05）.4. After1hpre-incubation of10μmol/L BIM with RPMVEC, the effects of10μg/ml LPS to theCav-1mRNA expression in RPMVEC in6h were obviously decreased（P <0.05）.Conclusion1. RPMVEC was successfully isolated, cultured and identified in vitro;2.We verified the expression of Cav-1in RPMVEC;3. The expression of Cav-1mRNA inRPMVEC was both LPS concentration and time dependent;4. PKC inhibitor BIM cansignificantly reduced the expression of Cav-1mRNA which induced by LPS.