Affect Of Fluid Shear Stress Intensity On Fractalkine Expression Level In Endothelial Cells

Objective:Atherosclerosis is the base of variety of cardiovascular disease andthe effect of shear stress which generated by blood flow on endothelial cellmorphology and function play an important role in cardiovascular diseases.Inendothelial cells,have found a variety of genes regulated by shearstress,including chemokines.Fractalkine(FKN) is a chemokine which is onlyCX3C subfamily members has been found,have both chemotaxis andadhension specificity,associated with variety of inflammatory diseases and invivo study found that the expression of FKN increased in the formation of ASplaques induced by low shear stress.A large number of studies have shownthat FKN has great significance for AS formation and development.Methods:1.To chose more suit investigate object, Injected0.1%collagenasetypeⅡ in fresh (get healthy cesarean section newborn umbilical cord, length15-20cm, the specimens time≤4hours) human umbilical vein getendothelial cells culture with M199growth medium （25%ECGS）,EA.HY926cell line culture with M199growth medium. Compared twogroups of, cell morphology under optical electron microscope,cellmorphology under electron microscope, Ⅷ factor-related antigenimmunohistochemical staining, cells expressing CD31antibody and growthcurve of two group of cells. Results:⑴Cell morphology under opticalelectron microscope：The primary HUVEC in vitro3~4d can be coveredwith single layer, growth seen was spindle-shaped cells, full,and vorter-likearrangement,after cell passage can be find lumen-like structure; EA.HY926cell lines has more cytoplasmic granules,2to3can be covered with singlelayers, can be grown to multiple layers.⑵Ⅷ factor-related antigenimmunohistochemical staining： To assess Ⅷ factor related antigenimmunohistochemical stainin by Image-Pro Plus6.0software,the score ofprimary HUVEC’s mean density was4138.8±594.01, EA.HY926cell line as2234.02±78.78, T test derived P <0.01, both expressed difference wasstatistically significant. Staining of primary HUVEC cells were significantlyhigher than EA.HY926cell lines.⑶Cell morphology under electronmicroscope:Through transmission electron microscope, in primary HUVECcan be found more cross-section and longitudinal section of moreWebel-Palade bodies, wherease in EA.HY926cell lines was less.⑷Cellsexpressing CD31antibody:The expression of CD31antibodies in primaryHUVEC cells (85.36%) higher than EA.HY926cell line (70.82%), T test forstatistical analysis, bilateral P <0.001, two cell CD31expression differencewas statistically significant, primary HUVEC higher than EA.HY926cellline.⑸Primary cells from48hours to enter the growing season, afterreaching a peak in96hours, the cells begin to aging, tend to apoptosis.EA.HY926cell lines48h to enter the growing season,96-120h peaked intothe stable growth and has a little extent of growth. Conclusion: Umbilicalvein digested with collagenase type Ⅱand M199growth medium canquickly obtain a large number of endothelial cells, its biologicalcharacteristics superior to HUVEC strains,however the EA.HY926cell linecharacterized by more cell numbers,simple culture methods and highsurvival rate, use it in more cell trauma and complex study.2.To investigatethe role of intensity of fluid shear stress on Fractalkine expression inEA.HY926human umbilical vein endothelial cell lines(HUVECs), we hadEA.HY926cell lines exposed to shear stress2.62,4.58,6.54,10.47,15.71，and19.64dyne/cm2respectively and employed quantitative reversaltranscription-polymerase chain reaction (qRT-PCR) to assay the expressionof FKN mRNA and employed quantitative sandwich enzyme-linkedimmunosorbent assay (ELI SA) to measure the FKN protein. Results: Thereal time PCR results show that FKN has very little expression with untreatedcituation，at0.01±0.00, when the strength of Shear stress at4.58dyne/cm2,the Fractalkine gene expression strongest, at1.92±0.22,and the single-factoranalysis of variance show that it has statistically significant amount of thedifference with the other shear stress FKN mRNA expression. In order toprove the correlation between the strength of the shear stress of and theexpression Fractalkine mRNA, two sets of correlation analysis, the results ofcorrelation r=-0.35, p>0.05, two sets of data have no correlation.Method ELISA results show that HUVECs untreated with fluid shear stress secretedFKN very little in culture media，at0.016±0.001. when the strength of Shearstress at4.58dyne/cm2the Fractalkine protein strongest at0.15±0.04andthe single-factor analysis of variance show that it has statistically significantamount of the difference with the other shear stress protein expression. Inorder to prove the correlation between the strength of the shear stress of andthe expression Fractalkine protein, two sets of correlation analysis, the resultsof correlation r=-0.33, p>0.05, two sets of data have nocorrelation.Conclusion: The results suggest that the best strength ofshear-induced Fractalkine is4.58dyne/cm2, based on this strength can befurther studied the relationship between the Fractalkine gene expression andthe role of time. The low shear stress could induces much more expression ofFractalkine mRNA, which plays probably an important role in thepathogenesis of inflammation andarteroatherosclerosis.