联合运用晚期糖基化终产物和同型半胱氨酸诱导人脐静脉内皮细胞血管细胞黏附因子1 mRNA表达及机制

The Effects of Advanced Glycation End Products and Homocystein on the Expression of Vascular Cell Adhesion Molecule-1 via NADPH Oxidase in Human Umbilical Vein Endothelial Cell

目的探讨联合运用晚期糖基化终产物和同型半胱氨酸对人脐静脉内皮细胞血管细胞黏附分子1表达的影响和氧化应激的机制。方法酶消化法获取人脐静脉内皮细胞。逆转录聚合酶链反应检测血管细胞黏附分子1基因的表达。活性氧检测试剂盒检测细胞内氧自由基的荧光信号强度。结果晚期糖基化终产物(10-4～10-1g/L)和同型半胱氨酸(1.35×10-3～1.35g/L)组呈浓度依赖性诱导人脐静脉内皮细胞血管细胞黏附分子1的表达,晚期糖基化终产物(10-4g/L)和同型半胱氨酸(1.35×10-3g/L)联合作用能进一步增加人脐静脉内皮细胞血管细胞黏附分子1基因的表达(1.09±0.18),与单独晚期糖基化终产物(0.14±0.07)和同型半胱氨酸组(0.18±0.06)相比其表达分别增加了7.78倍和6.05倍(P<0.01);还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶特异性抑制剂二亚苯基碘能显著抑制晚期糖基化终产物和同型半胱氨酸联合诱导的血管细胞黏附分子1基因的表达(0.20±0.09比1.19±0.23,P<0.01);晚期糖基化终产物组和同型半胱氨酸组均能增加人脐静脉内皮细胞内氧自由基的荧光信号强度,晚期糖基化终产物和同型半胱氨酸联合作用时,细胞内氧自由基的荧光信号强度水平进一步升高,且二亚苯基碘能明显抑制上述效应。结论晚期糖基化终产物和同型半胱氨酸能增加细胞内氧自由基的荧光信号水平,使血管细胞黏附分子1基因的表达上调,且两者存在协同效应;氧自由基水平的增加是导致血管细胞黏附分子1基因表达升高的重要因素;血管细胞黏附分子1可能是通过还原型烟酰胺腺嘌呤二核苷酸磷酸氧化酶途径实现的。

Aim To investigate the effects of advanced glycation end products（AGE）and homocystein（Hcy）on the expression of vascular cell adhesion molecule-1（VCAM-1）mRNA via oxidative stress mechanism in human umbilical vein endothelial cell（HUVEC）.Methods Collagenase was used to isolate the endothelial cell from human umbilical vein.RT-PCR was used to examine the expression of VCAM-1 mRNA.Inversion fluoescence microscope was used to observe the level of oxygen free radical.Results The expression of VCAM-1 mRNA was induced concentration-dependently by AGE（10-4～10-1 g/L）and Hcy（1.35×10-3～1.35 g/L）;AGE combined with Hcy group could promote the expression of VCAM-1 mRNA respectively 7.78 times and 6.05 times in comparison with AGE group and Hcy group（1.09±0.18 vs 0.14±0.07;1.09±0.18 vs 0.18±0.06,P0.01）;By comparison with AGE combined with Hcy group,the VCAM-1 expression of DPI group was obviously reduced（0.20±0.09 vs 1.19±0.23,P0.01）;In comparison with control group,AGE group and Hcy group could enhance oxygen free radical in HUVEC;The level of oxygen free radical in AGE combined with Hcy group was higher than that in AGE group and Hcy group;DPI could significantly inhibit the level of oxygen free radical.Conclusion AGE and Hcy can enhance the level of oxygen free radical in HUVEC and increase the VCAM-1 expression,and they have the cooperative effect;The enhancement of oxygen free radical is animportant factor on the expression of VCAM-1 mRNA;The above process is realized possibly through activating the NADPH oxidase.