The Effects Of Advanced Glycation End Products Andhomocystein On The Expression Of Vascular Adhesion Molecule-1mrna And Mechanism In Human Umbilical Vein Endothelial Cell

ObjectiveTo investigate the effects of advanced glycation end products (AGEs) and homocystein(Hcy) on the expression of vascular adhesion molecule-1(VCAM-1)mRNA via oxidative stress mechanism in human umbilical vein endothelial cell(HUVECs).MethodsCollagenase was used to isolate the endothelial cells from human umbilical vein. Human umbilical vein endothelial cells was identidied by Immunocytochemical stain technique and morphology observation under optical microscope.HUVECs were grouped as:①control: non-intervention factors;②AGEs group: the different concentration of AGEs and HUVECs were incubated for24h;③Hcy group: HUVECs and the different concentration of Hcy were incubated for 24h;④(AGEs+Hcy)group: HUVECs were incubated with AGEs and Hcy for 24h;⑤(AGEs+Hcy+DPI)group: HUVECs were first pretreated with DPI(10umol/L)for 30min,and incubated with AGEs and Hcy for 24h. RT-PCR was used to examine the expression of VCAM-1 mRNA in HUVECs.Inversion fluoescence microscope was used to observe the fluorescence intensity of ROS.ResultsIn the mixed suspension of single cells, most of which were Human umbilical vein endothelial cells. The identification of HUVECs was identified by CD34 and CD31 Immunocytochemical stain technique and morphology observation under optical microscope.The expression of VCAM-1mRNA was dose-dependently induced by AGEs and Hcy; AGEs combined with Hcy group could promote the expression of VCAM-1 mRNA respectively increased 7.78 times and 6.05 times in comparison with AGEs group and Hcy group(0.14±0.06 vs 1.09±0.18;0.18±0.05 vs 1.09±0.18,P<0.01); By comparison with AGEs combined with Hcy group,the VCAM-1 mRNA expression of DPI group was obviously reduced(0.20±0.09 vs 1.19±0.23,P<0.01).In comparison with control group, AGEs group and Hcy group could enhance ROS in HUVECS; The level of ROS in AGEs combined with Hcy group was higher than that in AGEs group and Hcy group;DPI could significantly inhibit the fluorescence intensity of ROS.ConclusionThe co-operative effect of AGEs and Hcy can enhance the fluorescence intensity of ROS in HUVECS and up-regulate the expression of VCAM-1mRNA; The enhancement of ROS is an important factor on the expression of VCAM-1 mRNA; The above process is realized possibly through activating the NADPH oxidase.