The Effect Of Secrete Of Vascular Endothelial Cell Stimulated By LPS And TG In Vitro

Objective: To utilize the optimized method of primary culture endothelial cells from human umbilical vein and form the cell model successfully in vitro . Based on this cell model, studying the effect of different concentration of lipopolysaccharide(LPS) and triglycerides(TG) on the expression of tumor necrosis factor (TNF) and endothelin(ET) , prove its transcriptional control mechanism at molecular level. Help to study the mechanism of heart disease and cerebral vessels disease, and to investigate the effect drugs of anti-endotoxins and drugs of preventing the heart disease and cerebral vessels disease. Method: 1. Compare the numbers of HUVEC that obtained by the perfusion method of different density of trypsin and collagenase injected into the human umbilical vein; compare the numbers of HUVEC that obtained by erosion and perfusion method ; compare the numbers of HUVEC that growth with culture fluid at different pH degree, then optimize the cell culture method. Identify the cells by using inverted microscope and by immunohistochemistry method. 2. Stimulate the cells by different concentration of lipopolysaccharide (LPS) and triglycerides (TG) ,collect the clear supernatant liquid at different time ,then calculate the expression of TNF and ET in the collection by radioimmunoassay (RIA).3. Synthetic primer of TNF and ET .A reverse transcription polymerase chain reaction (RT-PCR) method was used to amplify TNF, ET and internal control glyceradehyde phosphate hydrogenas mRNA. RT-PCR products were separated on 1% agarose gels. Gels were photographed and scanned, then the optical density of each band were determined by using Quantity One software. Results: 1 .Endothelial cells digested by 0.1% collagenase for 12 minutes ,culture the cells with RPMI1640 culture fluid at PH 7.4 degree , by using these methods cells growth better than other methods. 2.The cultured cells can be identified by using immunohistochemistry method. 3.A distinct increase of TNF and ET values...