The Study For Membrane Surface Ultrastructure Of Oxidative Stress Human Umbilical Vein Endothelial Cells By Atomic Forced Microscope

Objective: The purpose of the study was to find the rules and characteristics from dysfunction to structural changes on oxidative stress endothelial cells, and attempt to complete the structural repetition of intercellular adhesion molecule-1 (ICAM-1) on the endothelial cells surfaces. The oxidative stress model was established through a method of inducing cultured Human Umbilical Vein Endothelial Cell(HUVEC) to lipid per-oxidation by oxidized low-density lipoprotein(ox-LDL).Methods: HUVEC were isolated by 0.1% type I collagenase digestion for 15-20 min at 37℃, and cultured in low serum endothelial cell medium. After passage cultivation, we used the factor VIII related antibody to identify endothelial cell. Cells were used at passage 2 to 3. Passaged cells were seeded into culture plates or plastic dishes in a certain density. After 24 hours adherent growth, cells could reach 80% of fusion. A final concentration of 100μg/ml of ox-LDL cultured with HUVEC together. By the different induction time, cells were divided into 0 hour, 4 hour, 8 hour, and 16 hour four experimental groups. Each experimental group had a control group, which used PBS to instead of ox-LDL. MTT assay compared cell viability among four groups. Chemical assay detected NO in the supernatant from four groups cells to determine the functional status of HUVEC. AFM was applied to observe HUVEC surface ultrastructure for each group. We Selected 0h and 16h experimental groups for ICAM-1 immuno-gold labeling, then found gold particles and depicted the ICAM-1 molecule characterization in AFM.Results1 The number of live HUVEC in 8h experimental group was less than 0h experimental group(P<0.05), and live cells in 16h experimental group were further reduced than 0h, 4h, and 8h experimental groups(P<0.05). Cell viability in 16h control group was higher than the ones in 0h, 4h and 8h control groups (P<0.05). It showed a lower viability in ox-LDL experimental groups than PBS control groups during the same period after 8 hours induction(P<0.05).2 There were significant difference on NO content between 8h experimental group and 0h, 4h experimental groups(P<0.05), the former was less than latter; NO content in 16h experimental group was further reduced than 8h experimental group(P<0.05). However, there was no significant difference on NO content for each two control groups (P>0.05). It showed a lower NO content in ox-LDL experimental groups than PBS control groups during the same period after 8 hours induction(P<0.05).3 In 4h experimental group, HUVEC surface particles start to congregate with irregular distribution, large uplifts with higher height than 0h experimental group emerged; In 8h experimental group these changes present more obviously, even a few small holes appeared; In 16h experimental group, uplifts and hollows increased furtherly on number, size and depth than the other three experimental groups. There were significant differences on cell surface roughness(nm) for each two experimental groups (13.666±2.196 vs 15.904±2.203 vs 17.688±2.076 vs 21.609±1.867, P<0.05), while there was no significant difference on cell surface roughness(nm) for each two control groups (13.627±2.218 vs 13.659±2.183 vs 13.665±2.175 vs 13.974±2.478, P>0.05). It showed a higher surface roughness in ox-LDL experimental groups than PBS control groups during the same period after 4 hours induction(P<0.05).4 The gold particles in AFM Phase picture performed high light spots without tail. Their average diameter was 21.548±1.692 nm. And the second IgG performed round particles at the corresponding position in Height picture. Their average diameter was 50.944±4.404nm.5 In 0h experimental group, there was only few of high light spots in AFM Phase picture after immuno-gold labeling. Their average diameter was 53.826±5.367nm. It was different from the gold particle in pure colloidal gold IgG (P<0.01). Furthermore, there was no round particle at corresponding position in Height picture. In 16h experimental group, the number of high light spots in Phase picture increased significantly. Their average diameter was 21.355±1.905nm. It showed no difference from the gold particles in Phase picture of pure colloidal gold IgG (P>0.05). Furthermore, there were some round particles at corresponding positions in Height picture. Their average diameter was 51.610±4.752nm. It was not different from the round particle in Height picture of pure colloidal gold IgG, too(P>0.05).6 In the reconstructed 3D image of ultra-structure of immuno-gold treated HUVECs in 16h experimental group, we could see the hemispherical immuno-gold IgG. It was close to an uplift that seemed as peak. The average size of these uplifts was 170nm×220nm, while average height was 22±2.326nm.Conclusion1 HUVEC viability could be inhibited by ox-LDL. HUVEC function gradually diminished as induction time gone.2 Membrane surface ultra-structure of HUVEC experienced changes at early stage during the development of atherogenesis. It showed a time-dependent feature.3 ICAM-1 molecule was one of components in all the cell surface particles. ICAM-1 antigen-antibody complex on cells surfaces like peaks. Technique of AFM with immuno-gold labeling was feasible for cell surface component qualitative analysis and morphological description.