Experimental Study On The Relationship Between Heparin Sulfate Proteoglycans Of Endothelial Cells And Cell Adhesion

Objective Cellular adhesion is a very important biological phenomenon for multicellular biology in maintaining their architecture and function, and is also show communication relationships within cells, the action of cell adhesion is strong connect with various pathological or physiological processes. Vascular endothelial cell is the target cell for cell-adhesion in shock, wound, inflammation and tumour. The control mechanisms of cell adhesion are very complex involved many activated substances participated in the actions. The traditional adhesion molecules hold the dominant function to control the cell-adhesion actions. Recently, the heparin sulfate proteoglycans (HSPGs), which is mainly proteoglycans(PGs) anchor upon the surface of cells, has been studied hotly. The human umbilical vein endothelial cells (hUVECs) were taken as experimental cells after successfully sub-cultured in vitro. Whether the HSPGs can express stably on the cultured hUVECs was determined firstly. The contents of HSPGs on the surface of endothelial cells were measured under the exterior intervening factors action, and the effect of exterior intervening factors on HSPGs were evaluated. The cell adhesion models, in which the mononuclear apart from circulate blood in normal human body can adhere to hUVECs, were used to evaluate the relation between the HSPGs contents and cell adhesion. In order to simulating the pathological active of normal endothelial cells (ECs) in clinic leukaemia, the blood serums come from acute leukaemia were used to cultured ECs, and the RNA express and contents of HSPGs on ECs were determined. The cell adhesion models, in which the mononuclear can adhere to hUVECs that have been actived by leukemia serum beforehand, were used to determine the relation between the HSPGs contents and cell adhesion in leukaemia patients. The aims of study were to explore the new ideas or methods on preventing or treatment for extramedullary infiltration in leukaemia patients. Methods 1. Enzyme-digestion method was used to obtain hUVECs, and cell number can up to scratch for experiment study by sub-cultured in vitro. 2. The total ECs RNA were extracted, and the special primers for Syndecan-1 and Glypican-1 were synthesized based on the total gene sequences for two families of HSPGs on ECs in gene bank, human GAPDH primers were act as internal control, RT-PCR was performed to half-determine the expression of Syndecan-1, Glypican-1 on the surface of ECs. 3. NaClO3, 4-methylumbelliferyl-7-β-D-xyloside (4-β-DX), heparin, tumor necrosis factor α(TNFα), heparinaseⅢand leukaemia serum were taken as intervening factors to work on the ECs. The RT-PCR method was used to determine the expression of Syndecan-1 and Glypican-1 on the surface of ECs, and biocolor measure method was used to evaluate the PGs contents in the ECs. 4. The normal human mononuclear leukocyte aparted from peripheral blood by density centrifugation were co-cultured with ECs, on which the HSPGs contents have been modified by intervening factors. Methyl thiazolyl tetrazolium (MTT) colorimetry method was used to determine the mononuclear-endothelial cell adhesion, inverted microscopy and scanning electron microscopy were used to observe the cell adhesion. 5. The flow cytometry method was used to determine the ECs activated by exterior TNFαand leukaemia serum, the expression for CD62E antigen was evaluated to determine the effect of traditional adhesion molecular and confirm the function of HSPGs. Results 1. The cultured hUVECs can maintain finer growth station. Been confirmed by immunohistochemistry technique, the ECs can act as objected cell. 2. Based on the total gene sequences for HSPGs on ECs in gene bank, the special primers for Syndecan-1 and Glypican-1 were synthesized. The total ECs RNA extractions were amplified by RT-PCR and the sequence of HSPGs gene is coincident with gene bank. The results confirmed that Syndecan-1 and Glypican-1 can expression on the surface of ECs. 3. Different intervening factor affect HSPGs expression 1) The various inter...