上海交通大学学报（医学版）

›› 2011, Vol. 31 ›› Issue (7): 900-.doi: 10.3969/j.issn.1674-8115.2011.07.007

• 论著（基础研究） • 上一篇    下一篇

尿毒症患者血清对体外培养的血管内皮细胞的损伤作用

李 聪, 郝 旭, 周 桥, 卢 颖, 王伟铭, 陈 楠   

  1. 上海交通大学 医学院附属瑞金医院肾内科, 上海 200025
  • 出版日期:2011-07-28 发布日期:2011-07-27
  • 通讯作者: 陈 楠, 电子信箱: Chennan@medmail.com.cn。
  • 作者简介:李 聪（1984—）, 女, 硕士生；电子信箱: licong6521@163.com。
  • 基金资助:

    上海市科委重大课题（08dz1900502）

In vitro injury effects of sera of patients with uremia on vascular endothelial cells

LI Cong, HAO Xu, ZHOU Qiao, LU Ying, WANG Wei-ming, CHEN Nan   

  1. Department of Nephrology, Ruijin Hospital, Shanghai Jiaotong University School of Medicine, Shanghai 200025, China
  • Online:2011-07-28 Published:2011-07-27
  • Supported by:

    Shanghai Science and Technology Committee Foundation, 08dz1900502

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摘要：

目的 探讨尿毒症患者血清干预对体外培养的血管内皮细胞的作用及临床意义。方法 分别以尿毒症患者和正常健康者血清（体积分数均为25%）处理人脐静脉内皮细胞（HUVEC）（尿毒症血清组和正常血清组，n=10）24 h，设立空白对照组。光学显微镜观察细胞形态；流式细胞术检测细胞凋亡率；Real-Time PCR检测细胞内白介素8（IL-8）、内皮素1（ET-1）和内皮型一氧化氮合酶（eNOS）的mRNA表达；收集细胞培养上清液，酶联免疫ELISA法检测IL-8 和ET-1的质量浓度，硝酸盐还原法检测一氧化氮（NO）浓度。以不同体积分数（10%、15%、20%、25%）的尿毒症患者血清处理HUVEC 24 h，MTS比色法检测细胞增殖率。结果 与空白对照组和正常血清组比较，尿毒症血清组HUVEC的细胞数量明显减少，核固缩、边缘化，胞质浓缩；尿毒症血清组细胞凋亡率为（55.59±5.21）%，显著高于正常血清组的（12.20±6.20）%和空白对照组的（0.38±0.31）%（P<0.05）；与正常血清组比较，尿毒症血清组HUVEC中IL-8、ET-1 mRNA表达上调，eNOS mRNA表达下调；细胞培养上清中IL-8、ET-1的质量浓度升高，NO浓度降低(均P<0.05)。不同体积分数尿毒症血清处理HUVEC的细胞增殖率均显著低于相应正常血清组和空白对照组（P<0.05）。结论 尿毒症患者血清干预使HUVEC细胞形态发生改变，抑制细胞增殖并促进细胞凋亡，使细胞IL-8和ET-1分泌增加而NO分泌减少，导致血管内皮细胞功能受损。提示尿毒症患者长期处于慢性炎症状态，心血管疾病的发病风险较高。

关键词: 尿毒症, 血管内皮细胞, 白介素8, 内皮素1, 内皮型一氧化氮合酶

Abstract:

Objective To investigate the in vitro effects of sera of patients with uremia on vascular endothelial cells, and explore their clinical significance. Methods Human umbilical vascular endothelial cells (HUVEC) were treated with sera of patients with uremia (uremic serum group, n=10) and healthy controls (normal serum group, n=10)(volume fraction of 25% for both group) for 24 h, and blank control group was established. The cell morphology was observed by immunohistochemical staining, cell apoptotic rates were determined by flow cytometry, and Real-Time PCR was employed to detect the expression of interleukin-8 (IL-8), endothelin-1 (ET-1) and endothelial nitric oxide synthase (eNOS) mRNA in cells. The culture supernatant fluid was collected, the mass concentrations of IL-8 and ET-1 were measured by ELISA, and the concentration of nitric oxide (NO) was determined by nitrate reduction method. HUVEC were treated with sera of patients with uremia of different volume fractions (10%, 15%, 20% and 25%), and cell proliferation rates were determined by MTS method. Results The number of HUVEC in uremic serum group was smaller than those of blank control group and normal serum group, and condensed or fragmented nuclei and condensed cytoplasm were observed in uremic serum group. The cell apoptotic rate in uremic serum group [(55.59±5.21)%] was significantly higher than those in normal serum group [（12.20±6.20）%] and blank control group [（0.38±0.31）%] （P<0.05）. Compared with normal serum group, the expression of IL-8 and ET-1 mRNA in HUVEC increased, the expression of eNOS mRNA in HUVEC decreased, the mass concentrations of IL-8 and ET-1 in cell supernatant fluid increased, and the concentration of NO decreased in uremic serum group. The cell proliferation rates of HUVEC treated with uremic sera of different volume fractions were significantly lower than corresponding ones in normal serum group and blank control group （P<0.05）. Conclusion Treatment with sera of patients with uremia may change the morphology, inhibit the proliferation, promote the apoptosis, enhance the secretion of IL-8 and ET-1 and inhibit the secretion of eNOS and NO of HUVEC, and lead to the dysfunction of vascular endothelial cells. Patients with uremia are under chronic inflammatory state in a long term, and may have a greater risk of developing cardiovascular diseases.

Key words: uremia, vascular endothelial cell, interleukin-8, endothelin-1, endothelial nitric oxide synthetase