上海交通大学学报（医学版）

上海交通大学学报（医学版）

• 论著（基础研究） • 上一篇    下一篇

高糖及晚期糖基化终末产物环境对血管内皮细胞血管样结构形成的影响

林雪松1，汪 乐1，乔 亮2   

  1. 1.浙江省台州市中心医院烧伤科， 台州 318000； 2.上海交通大学 医学院附属瑞金医院灼伤整形科， 上海 200025
  • 出版日期:2014-05-28 发布日期:2014-05-30
  • 通讯作者: 乔 亮, 电子信箱: qiaoliang@yahoo.com。
  • 作者简介:林雪松（1970—）， 男， 副主任医师; 电子信箱: linxuesg@163.com。
  • 基金资助:

    国家自然科学基金（81272112）；台州市科学基金（091ky05）

Effects of high glucose and advanced glycation end products on formation of vascular-like structure of vascular endothelial cells

LIN Xue-song1, WANG Le1, QIAO Liang2   

  1. 1.Burn Center, Taizhou Central Hospital, Taizhou 318000, China； 2.Department of Burn and Plastic Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
  • Online:2014-05-28 Published:2014-05-30
  • Supported by:

    National Natural Science Foundation of China, 81272112; Science Foundation of Taizhou, 091ky05

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摘要：

目的 观察高浓度葡萄糖和晚期糖基化终末产物（AGEs）干预对体外培养血管内皮细胞的损伤后愈合、血管样结构形成能力和相关血管化调控因子表达的影响。方法 根据培养液中添加不同浓度的葡萄糖和AGES-BSA，将体外培养的人脐静脉内皮细胞（HUVECs）分为高糖组（30 mmol/L D-葡萄糖）、AGEs组（150 mg/L AGE-BSA）、高糖+AGEs组（30 mmol/L D-葡萄糖+150 mg/L AGE-BSA）和正常组（5 mmol/L D-葡萄糖），另设甘露醇组（30 mmol/L 甘露醇），电子细胞基质阻抗判断法（ECIS）测定HUVECs损伤模型增殖曲线，倒置显微镜下观察HUVECs在Matrigel基质中血管样结构的形成情况；ELISA法检测细胞培养上清液中血管内皮生长因子（VEGF）和血管生成素-2（Ang-2）的表达，荧光显微镜下观察HUVECs中血管生成素受体Tie2的表达。结果 ECIS法测定结果显示：各组HUVECs增殖曲线形态相似，反映愈合率的斜率无明显差别。倒置显微镜观察发现：高糖组、AGEs组和高糖+AGEs组形成血管样结构的长度显著小于正常组（P<0.05或P<0.01）。ELISA检测结果显示：与正常组比较，高糖组、AGEs组和高糖+AGEs组细胞培养上清液中Ang-2水平显著升高，VEGF水平显著降低（P<0.05）。荧光显微镜观察发现：Tie-2受体在正常组HUVECs的细胞膜和细胞质中均有表达，在AGEs组的HUVECs中仅表达于细胞核中。结论 在高糖和（或）AGEs环境下，血管内皮细胞血管样结构形成能力受到抑制，其机制可能与Ang-2表达上调、VEGF表达下调以及Tie-2受体在细胞内不同部位的差异性表达有关。

关键词: 葡萄糖, 晚期糖基化终末产物, 血管化, 血管内皮细胞

Abstract:

Objective To observe the effects of high concentration glucose and advanced glycation end products (AGEs) on wound closure, formation ability of vascular-like structure, and the expression of relevant regulatory factor of vascularization of vascular endothelial cells cultured in vitro. Methods Human umbilical vein endothelial cells (HUVECs) cultured in vitro were divided into the high concentration glucose group (30 mmol/L of D-glucose), AGEs group (150 mg/L of AGEs-BSA), high concentration glucose+AGEs group (30 mmol/L of D-glucose+150 mg/L of AGEs-BSA), normal group (5 mmol/L of D-glucose) according to different concentrations of glucose and AGEs-BSA added in the culture fluid, and the mannitol group (30 mmol/L of mannitol). The proliferation curve of injury model of HUVECs was detected by the electric cell-substrate impedance sensing (ECIS). The formation of vascular-like structure of HUVECs in Matrigel matrix was observed by the inverted microscope. The expressions of vascular endothelial growth factor (VEGF) and angiopoietin-2 (Ang-2) in cell culture supernatants were detected by the ELISA and the expression of Tie-2 receptor in HUVECs was observed by the fluorescence microscope. Results The results of ECIS showed that proliferation curves of HUVECs of each group were similar and differences of their slopes, which represented the healing rate, were not significant. Observations of the inverted microscope found that lengths of formed vascular-like structures of the high concentration glucose group, AGEs group, and high concentration glucose+AGEs group were significantly shorter than those of the normal group (P<0.05 or P<0.01). The results of ELISA indicated that expressions of the Ang-2 levels in cell culture supernatants of the high concentration glucose group, AGEs group, and high concentration glucose+AGEs group were significantly higher than those of the normal group and expressions of the VEGF levels were significantly lower (P<0.05). Observations of the fluorescence microscope showed that Tie-2 receptors were expressed in the cytomembrane and cytoplasm of HUVECs of the normal group and were only expressed in the nucleolus of HUVECs of the AGEs group. Conclusion High concentration glucose and/or AGEs can inhibit the formation ability of vascular-like structure of vascular endothelial cells. The mechanism may be relevant to the up-regulation of Ang-2, down-regulation of VEGF, and the differential expression of Tie-2 receptor in different locations of cells.

Key words: glucose, advanced glycation end product, angiogenesis, endothelial cell