氧化型低密度脂蛋白对人脐静脉内皮细胞功能及膜表面超微结构的影响

Effect of Oxidized Low Density Lipoprotein on Function and Membrane Surface Ultrastructure in Human Umbilical Vein Endothelial Cells

目的观察人脐静脉内皮细胞在发生脂质过氧化过程中细胞功能及超微结构改变的规律和特点。方法用100 mg/L氧化型低密度脂蛋白诱导人脐静脉内皮细胞作为实验组,以与氧化型低密度脂蛋白等体积的PBS替代作为对照组,两组均连续培养0、4、8及16 h;用MTT比色法测定并比较各组细胞的增殖状态,用硝酸还原法测定各组细胞上清液中一氧化氮含量,用原子力显微镜对比观察各组贴壁人脐静脉内皮细胞表面的超微结构。结果随着氧化型低密度脂蛋白诱导时间的延长,实验组人脐静脉内皮细胞的增殖和功能明显减低,细胞表面隆起逐渐增大,分布越来越不规则,并伴随有凹陷或空洞出现;对照组随着培养时间的延长细胞表面结构变化不大。实验组0、4、8及16 h粗糙度分别为13.666±2.196 nm、15.904±2.203 nm、17.688±2.076 nm及21.609±1.867 nm,亚组间两两相比均存在显著性差异(P<0.05);而对照组0、4、8及16 h粗糙度分别为13.627±2.218 nm、13.659±2.183 nm、13.665±2.175 nm及13.974±2.478 nm,亚组间两两相比无显著性差异(P>0.05);与对照组相比,实验组细胞培养4 h后膜表面粗糙度存在显著性差异(P<0.05)。结论内皮细胞在发生脂质过氧化过程中功能逐渐减退,且表面超微结构在较早期就发生了改变,并随时间推移呈现动态变化。

Aim To observe the changes of human umbilical vein endothelial cells（HUVEC） in function and membrane surface ultrastructure during the lipid peroxidation. Methods HUVEC were induced by 100 mg/L oxidized low density lipoprotein（ox-LDL） as experiment group,then incubated for 0 h,4 h,8 h and 16 h.HUVEC in the PBS as control group was processed likewise.Cell viability was measured by MTT assay.The functional status of HUVEC was determined by detecting content of nitric oxide（NO） in the cultured cell supernate with nitric acid reduction assay.Atomic force microscope（AFM） was used for observation to membrane surface ultrastructure of HUVEC. Results The research showed that the proliferation ability of HUVEC in the experimental group were inhibited,cell functions were attenuated,and the longer incubation,the more significantly effects.At the same time,the eminentias on cell surface became larger,distribution irregularity,even some caveolaes and holes appeared.However,there was no change in control groups.Cell surface roughness analysis showed that roughness on 0 h,4 h,8 h and 16 h experimental groups were 13.666±2.196 nm,15.904±2.203 nm,17.688±2.076 nm and 21.609±1.867 nm,respectively.There were significant differences between each two（P0.05）.In the comparison,the control group were 13.627±2.218 nm,13.659±2.183 nm,13.665±2.175 nm and 13.974±2.478 nm,respectively,and no significant differences between each two（P0.05）.Compared with the control group,roughness in the experimental groups after 4 hours＇ induction was significantly higher（P0.05）. Conclusions It was demonstrated that the cell function of endothelial cells were weakened and membrane surface ultrastructure changed in an early stage of lipid peroxidation,and perform a time-dependent rule.