尿皮素对体外培养的人脐静脉内皮细胞一氧化氮及一氧化氮合酶的影响

Effects of urocortin on levels of nitric oxide and nitric oxide synthase protein expression in cultured human umbilical vein endothelial cells

目的：通过检测尿皮素（UCN）对人脐静脉内皮细胞（HUVEC）一氧化氮（NO）生成及一氧化氮合酶（NOS）表达的影响,探讨UCN调节胎儿-胎盘血管张力的分子机制。方法：在离体培养的HUVEC中加入不同浓度的UCN（0、0.1、1、10nmol/L）、10nmol/L促肾上腺皮质激素释放激素（CRH）及向以上各组UCN/CRH同时加α-helical-CRH进行体外培养,用硝酸还原酶法检测细胞上清液中NO的水平,蛋白印迹法检测内皮型一氧化氮合酶（eNOS）和诱导型一氧化氮合酶（iNOS）蛋白表达水平的变化。结果：在与UCN共同培养4~8h后,HUVEC细胞上清液中NO水平呈时间和剂量依赖性变化,随着培养时间的延长,UCN浓度增加,NO水平逐渐升高;UCN组HUVEC iNOS表达水平比对照组明显升高（P〈0.05）,且呈浓度依赖性升高。而各组eNOS表达水平与对照组比较无明显变化（P〉0.05）。UCN/CRH＋α-helical-CRH组HUVEC上清液中NO水平和HU-VEC iNOS表达水平与对照组比较无明显变化,差异无统计学意义（P〉0.05）,与相同浓度UCN/CRH组比较NO水平和iNOS蛋白表达水平明显降低,差异有统计学意义（P〈0.05）。结论：UCN可促HUVEC合成及释放NO,NO生成增多与UCN通过促进肾上腺皮质激素释放激素2β受体（CRH-R2β）正相调节iNOS蛋白表达有关,这可能是UCN调节胎儿-胎盘血管张力的分子机制之一。

Objective：To observe the effects of urocortin on the levels of nitric oxide （NO） and the expression of inducible nitric oxide synthase （iNOS） and endothelial nitric oxide synthase （eNOS） protein in human umbilical vein endothelial cells （ HUVECs）, and further in- vestigate the molecular mechanism of urocortin dilatation of fetal-placental vessels. Methods： HUVECs were cultured respectively with different concentrations of urocortin （0, 0. 1,1, 10nmol/L）, 10nmol/L corticotropin-releasing hormone （CRH） and urocortin/CRH ＋ α-helicalCRH. The method of nitrate reductase was used to determine the contents in culture superna- tant. NOS protein expression was determined by Western blotting. Results：The NO contents in supematant were increased after coculturing with urocortin in HUVECs for 4 ~ 8hours,with the feature of time- and concentration-dependent increase, with a significant difference between the UCN/CRH groups and blank control group （ P 〈 0.05 ）. The expressions of iNOS in the cells with urocortin were increased compared with control group （P 〈 0.05 ）, with the feature of condent increase, but there was no difference between the expression of eNOS in the cells with urocortin and control group（ P 〉 0.05 ）. The NO contents and expressions of iNOS in the cells with urocortin ＋ α-helicalCRH were decreased compared with equal concentration of urocortin （P 〈 0.05 ）, but there was no difference compared with control group （ P 〉 0.05 ） Conclusion： Urocortin stimulates the release of NO from HUVECs, and the change of NO may be the result of iNOS 2βreceptor（ CRH-R2 tion of fetal-placental upregulation induced by urocortin through corticotropin-releasing hormone β）. This may be one of the molecular mechanisms of urocortin vasodilavessel.