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Effects of LPS and GLU on Secretion of t-PA and PAI-1 from HUVEC in Vitro

Author: ZhangXiaoYan
Tutor: LiangChao
School: Jiangxi Medical College
Course: Pathogen Biology
Keywords: Umbilical vein Endothelial Cells Separate Endotoxin Glucose Plasminogen activator inhibitor-1 Tissue-type plasminogen activator
CLC: R363
Type: Master's thesis
Year: 2005
Downloads: 37
Quote: 0
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Abstract


Objective: To investigate human umbilical vein endothelial cells (Human umbilical endothelial cell, HUVEC) Isolation, culture and identification methods in the establishment of in vitro vascular endothelial cells in primary culture model based on the observation of different concentrations of bacterial endotoxin ( Lipopolyssacarides LPS ) and glucose ( Glucose , GLU ) vascular endothelial cell tissue-type plasminogen activator (Tissue plasminogen activator, t-PA) and plasminogen activator inhibitor -1 (Plasminogen activator inhibitor-1, PAI-1) expression in provide experimental basis for further study of the mechanism of the formation of a variety of thrombotic diseases . Methods: No. 5 one-time Daibing vein infusion needle perfusion digestive juice , respectively, 0.125% , 0.25% trypsin and 0.1% collagenase digestion umbilical vein intima digest the results of the three enzyme solution , and in the inverted phase contrast microscope observed under digestion products morphological characteristics , resulting cells were identified with immunohistochemical method . By enzyme-linked immunosorbent assay ( ELISA ) to detect groups HUVEC culture medium PAI-1 and t-PA content . And reverse transcription polymerase chain reaction ( RT - PCR ) , reverse transcription and amplification of PAI-1 and an internal reference glyceraldehyde 3 - phosphate dehydrogenase ( GAPDH ) mRNA amplification product was purified by agarose gel electrophoresis , gel analysis software optical density value of each strip was measured . Results: 1. 0.125% , 0.25% trypsin and 0.1% collagenase digestion time for 15min, 10min, 12min. 2 inverted phase endothelial cells under the microscope as a monolayer , cobblestone mosaic arrangement . Immunohistochemistry was used to detect the memory cells in factor VIII -related antigen . 1μg/ml LPS can significantly promote HUVEC secretion of PAI - 1 , 2h began to rise up to maximum effect , 24h . 4. 5.5mmol/LGLU role HUVEC after various period of time no significant changes in the PAI-1 secretion . 40mmol/LGLU role HUVEC 24 h , PAI -1 protein secretion reached its peak , and significant increase in PAI-1 mRNA level . (5) the concentration of GLU and LPS to HUVEC.

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