外源性一氧化氮促进人脐静脉内皮细胞损伤修复的体外实验研究  

In vitro study of ectogenous nitric oxide on improving repair of human umbilical vein endothelia cell injury

作　　者：陈鸿强[1] 秦士勇[1] 李桂杰[1] 王明海[1] 张鑫[1] 张涛[1] 刘菲[1] 张曙光[1] Chen Hongqiang;Qin Shiyong;Li Guijie;Wang Minghai;Zhang Xin;Zhang Tao;Liu Fei;Zhang Shuguang(Department of Vascular Surgery,Qianfashan Hospital,Shandong University,Shandong Province,Jinan 250014,China)

机构地区：[1]山东大学附属山东省千佛山医院血管外一科,济南250014

出　　处：《中华血管外科杂志》2018年第2期108-113,共6页Chinese Journal of Vascular Surgery

基　　金：山东省医药卫生科技发展计划项目（2014WS0110）

摘　　要：目的观察外源性一氧化氮（NO）对体外培养的正常及损伤模型中的人脐静脉内皮细胞（HUVEC）的干预变化，并探讨其修复机制。方法建立HUVEC的损伤模型，利用外源性NO供体硝普钠（SNP）进行干预。实验分为三组：正常HUVEC组（正常组）；H2O2诱导HUVEC损伤模型组（损伤组）；SNP干预H2O2诱导HUVEC损伤模型组（干预组）。光镜观察细胞形态学变化、Hoechst33258蓝色荧光染色后观察凋亡小体；Western Blot法检测eNOS、Caspase-7蛋白表达水平；ELISA法检测VEGF水平及免疫组化法检测PCNA表达水平。结果细胞形态及凋亡小体观察：正常HUVEC在H2O2诱导下发生氧化应激反应性损伤，细胞形态发生改变，出现凋亡小体；干预组细胞形态基本正常，凋亡小体明显减少。Western Blot法检测：损伤组eNOS表达水平降低，干预组eNOS表达回升，接近正常组含量；正常组未见Caspase-7蛋白表达，损伤组可检测到明显表达，干预组表达量显著减少。ELISA法检测：干预组VEGF含量高于正常组和损伤组，与损伤组比较差异有统计学意义（P〈0.05）；免疫组化法检测：干预组PCNA表达[（9.87±0.75）%]，与正常组[（10.25±0.63）%]比较差异无统计学意义（P〉0.05），与损伤组[（3.04±0.34）%]比较差异有统计学意义（P〈0.05）。结论在一定剂量外源性NO干预下可提高内皮细胞中eNOS基因的自身合成，促进损伤内皮细胞形态的修复，抑制凋亡发生以及保护损伤内皮细胞的增殖、合成及分泌等重要功能。ObjectiveTo study the influences of ectogenous nitric oxide（NO） on morphologic and apoptotic bodies of in vitro cultured normal and injured human umbilical vein endothelial cell （HUVEC）, and to discuss the repair mechanism.MethodsWe built the injured HUVEC model, and assessed the intervention of ectogenous NO sodium nitroprusside on injured HUVEC. HUVEC was divided into three groups： normal group, injury group and intervention group. The morphological changes of the cells and apoptotic bodies after the Hoechst33258 blue fluorescence staining in the light microscope was observed. The expression of endothelial nitric oxide synthase （eNOS） and Caspase-7 was tested by Western blot, vascular endothelial growth factor （VEGF） by ELISA and proliferating cell nuclear antigen （PCNA） by immunohistochemistry.ResultsNormal HUVEC were injured by oxidative stress induced by H2O2, the cellular morphological change and apoptotic bodies was observed. The morphological change in intervention group was negligible, and apoptotic bodies decreased significantly. Expression of eNOS decreased in injury group, which recovered in intervened group. No expression of Caspase-7 in normal group was observed, as it could be detected in injury group, while it decreased significantly in intervention group. Level of VEGF as higher in intervention group than those in other two groups, with significant difference （P〈0.05） between intervention and injury groups. Expression of PCNA in intervention group [（9.87±0.75）%]was almost equal to that in normal group[（10.25±0.63）%],and was higher than that in injury group[（3.04±0.34）%]. There was a significant difference （P〈0.05） between intervention and injury groups.ConclusionsIntervention of a certain dose of ectogenous NO can up-regulate the expression of eNOS gene and eNOS self-synthesis, improve repairing of endothelial cells, resist apoptosis and protect function of proliferation, synthesis and excretion of endothelial cells.

关 键 词：一氧化氮 内皮细胞 损伤修复 

分 类 号：R[医药卫生]