摘要
目的以人脐静脉内皮细胞（HUVECs）作为体外模拟高血糖记忆的研究对象，观察蛋白激酶C（PKC）β2、活性氧（ROS）对其表达血管内皮生长因子（VEGF）、血管细胞黏附分子1（VCAM-1）mRNA的影响，并探讨PKCβ2、ROS在其中可能的交互作用。方法将培养的HUVECs分为以下5组：正常糖（NG，5mmol／LD-葡萄糖×3周）组、高糖（HG，25mmol／LD-葡萄糖×3周）组、记忆（M，25mmol／LD-葡萄糖×2周＋5mmol／LD-葡萄糖×1周）组、记忆PKCp，转染[MB，25mmol／LD-葡萄糖×2周＋5mmol／LD．葡萄糖×1周＋PKCp2重组腺病毒（Ad5-PKC[32）]组、记忆＋Ot-硫辛酸（MA，25mmol／LD-葡萄糖×2周＋5mmol／LD-葡萄糖×1周＋62．5μmol／LOt-硫辛酸）组。以RT—PCR法检测VEGF、VCAM．1mRNA表达水平，用荧光显微镜和荧光酶标仪测定ROS水平，采用激光共聚焦检测PKCβ2蛋白的表达和转位。结果（1）HG组和M组VEGF、VCAM-1mRNA表达增加，分别为NG组的1．76倍、1．35倍和1．69倍、1．43倍（P值均〈0．05）。（2）HG组和M组ROS水平分别较NG组升高了86％、80％（P值均〈0．05）；MA组ROS水平较M组下降了38％，同时MA组VEGF、VCAM-1mRNA表达亦较M组显著下调，分别为M组的67％、78％（P值均〈0．05）。（3）HG组和M组PKCβ2核转位激活，定量分析显示，胞质／胞核荧光强度比值均较NG组下降，分别为NG组的70％、74％（P值均〈0．05）；MB组PKCβ2核转位较M组更为明显，胞质／胞核荧光强度比值为M组的77％，同时MB组VEGF、VCAM-1mRNA表达亦较M组进G 步上调，分别为M组的1．29倍、1．18倍（P值均〈0．05）。（4）MA组PKCβ2核转位较M组减少，胞质／胞核荧光强度比值为M组的1．18倍；而MB组ROS水平较M组上调了25％（P值均〈0．05）。结论HUVECs存在高糖记忆效应，PKCβ2-ROS交互环可能介导了这个效应的发生。
Objective To investigate the roles of protein kinase C （PKC）β2 and reactive oxygen species （ROS） in the mRNA expression of vascular endothelial growth factor （VEGF） and vascular cell adhesion molecule-1 （VCAM-I） in human umbilical vein endothelial cells （HUVECs）, imitating ＇ hyperglycemic memory＇ in vitro and the relationship between them. Methods The HUVECs were divided into 5 groups： normal glucose （NG, D-glucose 5 mmol/L ×3 weeks） group, high glucose （HG, D-glucose 25 mmol/L ×3 weeks） group, memory （ M, 25 mmol/L D-glucose × 2 weeks ＋ 5 mmol/L D-glucose × 1 weeks） group, memory transfected with PKCβ2 （ MB, M ＋ AdS-PKCβ2 ） group, memory plus α-lipoic acid （MA, M ＋62. 5 μmol/L α-lipoic acid） group. The mRNA expression of VEGF and VCAM-1 was detected with RT-PCR. ROS levels was tested with fluorescence microscopy and fluorescence microplate reader. The expression and translocation of PKCβ2 protein was observed with confocal laser scanning microscopy. Results （ 1 ） VEGF and VCAM-1 mRNA expression in group HG and M increased, being 1.76, 1.35 and 1.69, 1.43 folds of those in group NG （ all P 〈 0. 05 ）. （ 2 ） ROS levels in group HG and M increased 86% and80% as compared with those in group NG （ both P 〈 0.05 ）. As compared with group M, ROS levels in group MA decreased 38%, and VEGF,VCAM-1 mRNA expression in group MA decreased significantly to 67% and 78%. （3） PKCβ2 was activated and translocated from cytosol to nucleus in group HG and group M. Quantitative analysis showed the ratio of plasma/nuclear fluorescence intensity in group HG and M decreased to 70% and 74% of those in group NG （both P 〈 0. 05 ）. As compared with group M, PKCβ2 in group MB transloeated more obviously and the mRNA expression of VEGF and VCAM-1 in group MB significantly increased 1.29 and 1.18 folds of those in group M （both P 〈0. 05）. （4）As compared with group M ,the activation of PKCβ2 in group MA was inhibited and the ratio of plasma/nu
出处
《中华内科杂志》
CAS
CSCD
北大核心
2010年第3期239-244,共6页
Chinese Journal of Internal Medicine
基金
基金项目：国家自然科学基金（30570877）
关键词
蛋白激酶C
活性氧
内皮细胞
血糖
代谢记忆
Protein kinase C
Reactive oxygen species
Endothelial cells
Blood glucose
Metabolic memory
