重度子痫前期患者血清对人脐静脉血管内皮细胞的氧化性损伤研究

Oxidative damage effect of the serum of severe preeclamptic patients on human umbilical veinendothelial cell

目的探讨重度子痫前期患者血清对人脐静脉血管内皮细胞（HUVEC）造成的氧化性损伤。方法（1）HUVEC进行如下分组处理：20％健康孕妇血清刺激（正常血清组）、20％重度子痫前期患者血清刺激（PE组）、20％重度子痫前期患者血清＋3×10^3U／ml过氧化氢酶共同刺激（PE＋Cat组），并设置空白对照组，继续培养后，透射电镜观察细胞损伤情况，并用流式细胞仪检测凋亡相关蛋白AP02．7的表达情况。（2）激光共聚焦显微镜下，对HUVEC进行实时扫描的同时给予如下分组处理：正常血清组、PE组、PE＋Cat组、加入100μmo]／LH2O2（阳性对照组），分别检测胞内活性氧簇（ROS）、Ca^2＋及线粒体膜电位水平。结果（1）透射电镜下，PE组HUVEC的损伤明显，出现细胞膜脱落，并包裹细胞质形成自噬体，线粒体水肿，核固缩；但PE＋Cat组的HUVEC虽然线粒体有轻度水肿表现，但嵴清晰可见，细胞核形态基本正常，细胞损伤明显改善。PE组的HUVEC中AP02．7的表达率为（37．8±1．1）％，与空白对照组[（13．4±1．1）％]、正常血清组[（13．5±1．5）％]比较显著升高，差异均有统计学意义（P〈0．01）；而PE＋Cat组AP02．7表达率为（19．2±1．6）％，显著降低，与PE组比较，差异有统计学意义（P〈0．01）。（2）激光共聚焦显微镜下PE组的HUVEC胞内ROS、Ca^2＋荧光强度变化幅度分别为12．0±1．3、4．1±0．7，而正常血清组分别为1．1±0．4、0．6±0．4，PE＋Cat组分别为1．5±0．5、0．9±0．5，PE组分别与正常血清组、PE＋Cat组比较，差异均有统计学意义（P〈0．01）。PE组的HUVEC线粒体膜电位荧光强度在扫描120S时有显著降低，而正常血清组、PE＋Cat组变化不明显。结论重度子痫前期患者血清通过促进ROS生成，增强Ca^2＋超载水平和降低线粒体膜电位对血管内皮细胞造成氧化性损伤。

Objective To investigate oxidative damage effect of the serum of severe preeclamptic patients on human umbilical vein endothelial cells （HUVEC）. Methods （1） HUVEC were randomly divided into 4 groups according to the following ： blank group as control, normal group added 200/0 normal sera of pregnant women, group PE added 20% sera of severe preeclamptic patients, and group PE ＋ Cat added 20% sera of severe preeelamptic patients plus 3 ×10^3U/ml eatalase. After cultured for 24 hours, the injury morphology and APO2.7 expression of HUVEC were detected by transmission electron microscopy and flow cytometry respectively. （2） Under the real-time scanning by laser scanning eonfocal microscopy, HUVEC were randomly divided into 4 groups according to the following： control group added 100 μmol/L H202 as positive control, normal group, group PE, and group PE ＋ Cat. HUVEC of each group was scanned for 120 seconds to determine levels of reactive oxidative species （ROS）, calcium homeostasis, and mitoehondria membrane potential. Results （1） Obvious injury morphology of HUVEC was observed in group PE, and it was obviously improved by catalase in group PE ＋ Cat. Percentage of HUVEC expressed APO2. 7 was （37.8 ± 1.1 ） % in group PE, which was significantly higher than （ 13.4 ± 1.1 ） % in blankgroup or （ 13.5 ± 1.5 ） % in normal group, but significantly lower than （ 19.2 ± 1.6 ） % in group PE ＋ Cat （ all P 〈 0. 01 ）. （ 2 ） The fluorescence intensity curves of intracellular ROS and calcium showed slowly rising in group PE, hut no obvious changes in normal group and PE ＋ Cat. The values of ROS and calcium in group PE （12.0±1.3, 4. 1 ±0.7） were higher than those in normal group （1.1 ±0.4, 0.6 ±0.4）, but lower than those in group PE＋Cat （1.5±0.5, 0.9±0.5; allP〈0.01）.Conelusion The serum of severe preeclamptic patients caused oxidative damage on HUVEC by increasing intracellular ROS generation, calcium overload, and decreasing mitochondrial membrane Dotential.