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目的观察Exendin-4(Ex-4)对猪髋动脉内皮细胞(pig iliac endothelium cell lines,PIEC)释放一氧化氮(NO)的影响,及其与细胞内外钙浓度的关系。方法免疫荧光法检测确认PIEC上存在胰高血糖素样肽-1受体(glucagon-likepeptide-1,GLP-1R);Griess重氮化反应检测Ex-4对有钙、无钙2种培养基条件下内皮细胞NO释放的影响,流式细胞仪检测细胞内钙浓度,评价Ex-4诱导PIEC释放NO的时效-量效关系。给予内皮型一氧化氮合成酶(eNOS)信号阻断剂左旋-硝基精氨酸甲脂(L-NAME)预处理后,观察Ex-4对PIEC释放NO的影响。结果 PIEC上有GLP-1受体表达。Ex-4能刺激PIEC释放NO,作用有剂量及时间依赖关系,同时细胞内钙离子浓度也有升高[(62.67±1.53)vs(57.36±1.42),P<0.01]。无钙培养液组PIEC释放NO较有钙培养液组升高更明显[(2.014±1.040)μmol/L vs(1.624±0.424)μmol/L,P=0.03]。Ex-4刺激PIEC释放NO L-NAME预处理后,Ex-4诱导的PIEC释放NO减少(P<0.01)。结论 Ex-4通过激活eNOS增加内皮细胞NO释放,其eNOS激活作用可能与细胞内外钙离子变化相关。
Objective To investigate the effect of Exendin-4 (Ex-4) on the release of nitric oxide (NO) in pig iliac endothelium cell lines (PIEC) and its relationship with intracellular calcium concentration. Methods Glucagon-likepeptide-1 (GLP-1R) was detected on the PIEC by immunofluorescence assay. Griess diazotization assay was performed on Ex-4 cells with and without calcium The effect of Ex-4 on the release of NO from endothelial cells was evaluated by flow cytometry (FCM). After pretreatment with L-NAME, a signal inhibitor of endothelial nitric oxide synthase (eNOS), the effects of Ex-4 on PI-released NO were observed. Results There was GLP-1 receptor expression on PIEC. Ex-4 stimulated the release of NO by PIEC in a time-and dose-dependent manner, with an increase in intracellular calcium concentration (62.67 ± 1.53 vs 57.36 ± 1.42, P <0.01). The NO release from PIEC in calcium-free medium was significantly higher than that in calcium-supplemented medium [(2.014 ± 1.040) μmol / L vs (1.624 ± 0.424) μmol / L, P = 0.03]. Ex-4-induced PIEC release NO L-NAME pretreatment, Ex-4-induced PIEC release of NO decreased (P <0.01). Conclusion Ex-4 increases the release of NO from endothelial cells by activating eNOS. The activation of eNOS may be related to intracellular calcium changes.