Effect Of TNF-α On Expression Of Src-suppressed C Kinase Substrate In Rat Pulmonary Microvascular Endothelial Cell

Objective:To investigate effect of tumor necrosis factor-α(TNF-α) on expression of Src-suppressed C kinase substrate(SSeCKS) in rat pulmonary microvascular endothelial cell(PMVEC) and the interferring action of methylprednisolone sodium succinate on TNF-αinduced permeability injury of rat PMVEC monolayer and expression of SSeCKS.Methods:Cultured rat PMVEC were randomly divided into two groups.In time-dependent group,cells were cultured with TNF-α(5000 U/ml) for 0.5 h,1.5 h,3 h,6 h,12 h,respectively;in dose-dependent group,cells were cultured with TNF-α(200 U/ml,1000 U/ml,5000 U/ml) for 3 h.The interfering action of medicine:cells were cultured with TNF-α(5000 U/ml) and methylprednisolone sodium succinate(0.2 mg/ml) +TNF-α(5000 U/ml) for 1.5 h.The expression of SSeCKS was detected by RT-PCR and Western-blot analysis.All of these compared with normal control.Micro-infiltrator was used to measure the changes of rat PMVEC monolayer permeability coefficient(Kf) after exposure to TNF-α(5000 U/ml) and methylprednisolone sodium succinate(0.2 mg/ml)+TNF-α(5000 U/ml).All of these compared with normal PMVEC monolayer permeability.Results:1.We succeeded in isolating and culturing rat PMVEC.We confirmed that the cell was PMVEC,through the method of morphology and the binding experiment of FITC-PHA.2.We observed the specificity SSeCKS strap,through detecting normal rat PMVEC by RT-PCR and Western-blot analysis.3.The expression of SSeCKS was on a low level,normally.After incubation with TNF-α(5000 U/ml),the expression of SSeCKS mRNA and protein started to increase after 0.5h,reached the peak at 3h,and then decreased down gently but retained high expression to 12h.The expression of SSeCKS mRNA and protein at different times was significantly higher than normal control.It indicated that TNF-αupregulated SSeCKS mRNA and protein expression in a time dependent manner.4.After incubation with different concentrations of TNF-α,200 U/ml,1000 U/ml,5000 U/ml,the expression of SSeCKS mRNA and protein increased one by one.The expression of SSeCKS mRNA and protein at different concentrations was significantly higher than normal control.It indicated that TNF-αupregulated SSeCKS mRNA and protein expression in concentration dependent manner.5.The expression of SSeCKS protein of methylprednisolone sodium succinate+TNF-αgroup was significantly lower than TNF-αgroup.Meanwhile,the rat PMVEC monolayer permeability coefficient(Kf) was also significantly lower than TNF-αgroup.It indicated that methylprednisolone sodium succinate inhibited the permeability injury of rat PMVEC monolayer induced by TNF-α,coincident with down regulation of high SSeCKS protein expression.Conclusion:1.We succeed in isolating and culturing rat PMVEC from Wistar rat and indentified that was rat PMVEC.2.SSeCKS mRNA and protein expression were detected in rat PMVEC.3.TNF-αupregulated SSeCKS mRNA and protein expression in a time and concentration dependent manner.4.Methylprednisolone sodium succinate inhibited the permeability injury of rat PMVEC monolayer induced by TNF-α,coincident with down regulation of high SSeCKS protein expression,suggesting that SSeCKS plays an important role in permeability injury of rat PMVEC monolayer induced by TNF-α.It could regulate permeability of rat PMVEC monolayer.