脂氧素A4对缺氧损伤的人脐静脉内皮细胞的保护作用

Protective effects and its mechanism of lipoxins on human umbilical vein endothelial cells underhypoxia

目的探讨脂氧素A4（LXA4）对缺氧损伤的人脐静脉内皮细胞（HUVEC）的保护作用及其机制。方法体外培养HUVEC，培养后的细胞分别以单纯M199培养基培养（对照组）、氯化钻（CoCl2）诱导细胞缺氧（缺氧组）、不同浓度LXA4（1、10、100nmol/L）加入缺氧组细胞培养液中（药物干预组）；倒置相差显微镜下观察各组HUVEC的形态学变化；四甲基偶氮唑蓝（MTT）比色法检测不同浓度LXA4培养24h和100nmol/L的LXA4作用不同时间（4、8、12、24h）后缺氧HUVEC存活率；免疫细胞化学法检测细胞胞质内第Ⅷ因子相关抗原抗体（vWF）水平变化（以灰度值表示），细胞质内出现棕黄色颗粒为阳性；激光共聚焦显微镜观察细胞质内游离Ca^2＋荧光强度变化。结果（1）细胞形态：缺氧组HUVEC明显失去原有正常细胞形态，细胞变圆，核固缩；药物干预组正常细胞数量增多，加入100nmol/L浓度的LXA4后，大部分细胞形态与正常细胞形态基本相似。（2）细胞存活率：缺氧组细胞存活率为（40．1±3．9）％，药物干预组细胞培养液中加入1、10、100nmoVL浓度的LXA4后，细胞存活率分别为（52．9±1．4）％、（64．1±3．3）％、（76．6±1．6）％，分别与缺氧组比较，差异均有统计学意义（P〈0．05）。药物干预组细胞培养液中LXA。浓度为100nmo／／L时，作用4、8、12、24h，细胞存活率分别为（68．2±2．3）％、（82．5±1．4）％、（82．9±1．4）％和（72．2±8．5）％，且在12h时达峰值；药物干预组各时间点细胞存活率分别与缺氧组比较，差异均有统计学意义（P〈0．05）。（3）vWF水平：随着药物干预组细胞培养液中LXA。浓度的增加，其胞质内vwF水平逐步降低，LXA4浓度为1、10、100nmol／L时，vWF水平分别为203．9±0．7、204．6±0．9、191．8±0．5，分别与缺氧组比较，差异均有统计学意义（P〈0．05）。（4）Ca2’荧光强度：激光共聚焦显微镜观察结果显示，LXA4可提高缺氧HUVEC胞质内Ca^2＋荧光强度。结论LXA4对缺氧HUVEC维持正常的细胞形态起重要作用，并可提高其细胞存活率及降低细胞质内vWF水平，其保护机制可能是通过降低细胞内钙超载和转移细胞核内Ca^2＋使细胞质内Ca^2＋增加。

Objective To investigate protective effects and mechanisms of lipoxinA4 （ LXA4 ） on human umbilical vein endothelial cells （HUVEC） under hypoxia in vitro. Methods The HUVEC culture were divided into groups as followed： added M199 culture medium as normal control groups, added CoCl2 to mimic hypoxia in vitro as hypoxia group and added different concentrations of LXA4 （ 1,10,100 nmol/L ） were added to the induced hypoxial HUVEC as agents intervention group. Morphological changes of HUVEC were observed by using inverted phase contrast microscope. The influence of LXA4 on cell survival was investigated by methyl thiazolyl tetrazolium （MTF） assaying method after the treatment with different concentrations of LXA4 and 100 nmol/L lipoxinA4 according to different time （4, 8, 12 and 24 hours）. Theexpression of von-willebrand factor （vWF） was detected by immunocytochemistry method. The changes of cytosolic Ca^2 ＋ were measured by laser scanning confocal microscope. Results （ 1 ） Morphological changes ： the cells under hypoxia lost its normal shapes and showed necrosis, while the cells cocultured with 100 nmol/L LXA4 were normal appropriately. （2） Survival rate. the survival rates of HUVEC under hypoxia was （40. 1 ±3.9）% and increased to （52. 9±1.4）%, （64. 1±3.3）%, （76. 6 ± 1.6）% respectively when added with LXA4 with concentration of 1, 10, 100 nmol/L into culture medium. There was significant different survival rate when compared with that of hypoxia group. （3） The level of vWF ： The expression of vWF was decreased with the increasing concentrations of LXA4 added into culture medium, the gray values were 203.9 ±0.7 in 1 nmol/L,204.6±0. 9 in 10 nmol/L,191.8±0. 5 in 100 nmol/L respectively, which reached statistical difference in comparison with that of hypoxia groups （ P 〈 0. 05 ）. （ 4 ） Confocal analysis ：the intracellular free Ca^2＋ concentrations of HUVEC were intensified with LXA4 treatment. Conclusions LXA4 plays an important role in keeping the normal shape of HUVEC under hypoxia, can enhance survival of hypoxial HUVEC and decrease the level of vWF in cytoplasm. The protective mechanism might be via decreasing mitochondria Ca^2＋ overload and increasing cytoplasm Ca^2 ＋ by nucleus Ca^2＋ transference.