Effects Of LPS And GLU On Secretion Of T-PA And PAI-1 From HUVEC In Vitro

Objective: To investigate the primary culture method of endothelial cells from human umbilical vein ,study the effect of different concentration of lipopolysacch-aride(LPS) and glucose (GLU) on the expression of tissue plasminogen activator (t-PA) and plasminogen activator inhibitor type-1 (PAI-1),and help to study the mechanism of thrombus disease . Methods: 0.125%,0.25% trpsin and 0.1% collagenase were respectively injected into the human umbilical vein by using disposable vein transfusion needle No.5,after which the human umbilical vein endothelial cell(HUVEC) were indentified by morphology and immunohisto-chemistry under inverted microscope. PAI-1 and t-PA were measured in the media of HUVEC by a specific enzyme-linked immunosorbent assay (ELISA).A reverse transcription polymerse chain reaction method(RT-PCR) was used to amplify t-PA,PAI-1 and internal control glyceradehyde phosphate ehydrogenas(GAPDH) mRNA. RT-PCR products were separated on 1.5% agarose gels.Gels were photographed and scanned,then the optical density of each band were determined by using Quantity One? software. Results: 1. The optimized time for 0.125% trypsin,0.25% trypsin and 0.1% collagenase to digest the endothelial cells on lining of the human umbilical vein is 15min,10min and 12min respectively. 2. The cultured cells had a cobblestone appearance with a strict monolayer growth and had factor Ⅷ-related antigen.3. A distinct increase of PAI-1 values was found after LPS stimulation with a dose of 1ug/ml, starting at 2 hours. Maximum values were obtained at 24 hours. 4. No changes of PAI-1 values were observed after GLU stimulation with a dose of 5.5mmol/L. Maximum values of PAI-1 was found after GLU stimulation with a dose of 40mmol/L for 24 hours and a significant increase of PAI-1mRNA was also observed. 5. No differences in t-PA antigen were observed after either LPS or GLU stimulation.6. An increase of PAI-1 antigen was found in the media of HUVEC after stimulation with 1μg/ml of LPS and different doses of GLU,starting at 2 hours. Maximum values were obtained at 24 hours after stimulation with 1μg/ml of LPS and 40mmol/L GLU.And a progressive increase in the PAI-1mRNA was also obsearved at 24 hours after stimulation with 1μg/ml of LPS and different doses of GLU. 7.Whereas no differences in t-PA values were observed in the media of HUVEC after stimulation with 1μg/ml of LPS and different doses of GLU. Conclusion: 1. 0.1% collagenase was a better option to isolate endothelial cells from human umbilical vein."Irrigative digestion"technique was more practical and HUVECs were less contaminated during the process of digestion by using disposable vein transfusion needle No.5. HUVEC model was established successfully. 2. LPS and GLU can decrease fibrinolytic activity on the surface of HUVEC by inducing high expression of PAI-1mRNA without changing t-PA antigen, thus promote thrombus formation and development of atherosclerosis(AS). 3. PAI-1 may be the common inflammation factor of infection,diabetes mellitus and thrombus disease. 4. It is medically significant to monitor the dynamic secretion of PAI-1 with simultaneous detection of the concentration of LPS and GLU in the patients in order to predict the formation of thrombus and occurrence of the disorders associated with blood coagulation.