Induced Fgl2 Prothrombinase Expression In Rat Cardiac Microvascular Endothelial Cells By TNFα

PartⅠIsolation and culture of rats cardiac microvascular endothelial cells AbstractObjectives To explore a simple and convenient method of primary culture of the Rat cardiac microvascular endothelial cells(RCMECs)Methods Mechanical separation and two-step enzyme digestion with one-step gradient centrifugation were used to isolate cardiac microvascular endothelial cells. RCMEC were purified by filtration and difference adherent method. RCMECs were identified by the phase contrast microscopy and Fluorescence immunohistochemistry assay to dective factorⅧrelated antigen .Results RCMECs proliferate after 24h。RCMECs were identified according to the typical"cobblestone"morphology exhibited by confluent monolayer and factorⅧrelated antigen positive, the purity was about 90 %.Conclusions we established a simple culture system to isolate and culture RCMECs in vitro, which provides a powerful tool to study the mechanism of microthrombus. PartⅡInduced fgl2 prothrombinase expression in Rat Cardiac microvascular endothelial cells by TNFαAbstractObjectives To examine the effects of TNFα(Tumor necrosis factor alpha), which was present in the microenvironment of myocardio, on the fg12 prothrombinase expression and coagulation in rats cardiac microvascular endothelial cells in vitro.Methods Fluorescence immunohistochemistry and Western blot were used to the detection of fgl2 protein expression under the neutral and cytokine TNFαchallenged conditions, and the procoagulant activity prior and post the stimulation were detected by one-stage clotting assay.Results Fluorescence immunohistochemistry and Western blot suggested that TNF-αsignificantly up-regulated fgl2 expression at protein levels. 12.5ng/ml and 25ng/ml TNFαincreased the procoagulant activity (PCA) in rat cardiac microvascular endothelial cells, compare to the control(1.4527±0.186 vs -0.170±0.038和4.78±0.070 vs -0.170±0.038,P<0.05).Conclusions TNFαup-regulated fg12 protein expression in REMECs, depand on the level of TNFα, which in turn directly activate thrombinase. The later may correlated with the microcirculation dysfunction in cardiac microcirculation of inflammation disease accompanied by increased TNFα.