【作者】 闫立萍；

【导师】 刘静；

【作者基本信息】 兰州大学 ， 内科学， 2010， 硕士

【摘要】 目的：探讨内吗啡肽(EMs)对糖基化终末产物(AGEs)培养条件下人脐静脉内皮细胞(HUVECs)合成和分泌血管活性物质的影响。方法：从新鲜脐带中分离培养鉴定HUVECs,运用体外制备的糖基化修饰的牛血清白蛋白(AGEs-BSA)干预HUVECs,并进一步用不同浓度EMs(10μmol／ml,1μmol／ml,0.1μmol／ml,10nmol／ml)与AGEs-BSA共同干预HUVECs,同时设只加牛血清白蛋白(BSA)的HUVECs作阴性对照组；四甲基偶氮唑蓝(MTT)比色法检测HUVEC存活率,优化EMs的最佳作用浓度；硝酸盐还原酶法检测一氧化氮(NO)的浓度,分光光度法检测内皮型、诱导型一氧化氮合酶(eNOS、iNOS)的浓度,酶联免疫吸附(ELISA)法检测内皮素1(ET-1)的浓度,逆转录聚合酶链式反应(RT-PCR)检测eNOS、ET-1 mRNA的表达,免疫荧光法检测p38丝裂原活化蛋白激酶(p38MAPK)的表达。结果：AGEs-BSA可使HUVECs活力降低,并呈现时间依赖性,与正常对照组比较差异有统计学意义(P<0.01),时间依赖地刺激HUVECs分泌iNOS、NO、ET-1及上调ET-1 mRNA的表达,抑制eNOS分泌及下调eNOS mRNA的表达(P<0.05),并激活p38MAPK,使其核内表达的荧光值增高；EMs各浓度预处理组均可使AGEs-BSA作用下HUVECs存活率增加,NO分泌减少(P<0.05),iNOS、ET-1产生及ET-1 mRNA表达降低(P<0.05),eNOS分泌及eNOS mRNA表达增加(P<0.05),并呈时间依赖性和浓度依赖性；EMs可使AGEs-BSA作用下p38MAPK核内表达的荧光值降低。结论：AGEs-BSA可诱导人脐静脉内皮细胞活性下降及分泌内皮活性因子NO、eNOS、iNOS、ET-1受损；EMs对AGEs-BSA所致人脐静脉内皮细胞功能损伤有保护作用,并呈时间依赖性和浓度依赖性；EMs可抑制AGEs-BSA激活的p38MAPK的核内表达。更多还原

【Abstract】 Objective:To investigate effects of EMs on synthesis and secretion of vasoactive substances by human umbilical vein endothelial cells (HUVECs) in the condition of advanced glycation end products (AGEs).Methods:HUVECs were isolated from freshly collected umbilical cords, stimulated with AGEs-bovine serum albumin (AGEs-BSA), bovine serum albumin (BSA) or both AGEs-BSA and EMs, respectively. Then, HUVEC survival rate was calculated by MTT assay, the levels of NO, endothelial nitric oxide synthase and inducible nitric oxide synthase (eNOS, iNOS) were detected by colorimetric analysis, contents of endothelin-1 (ET-1) were detected by ELISA. Furthermore, expression of eNOS, ET-1 mRNA was measured by reverse transcription polymerase chain reaction (RT-PCR). In addition, expression of p38 mitogen-activated protein kinase (p38MAPK) was detected by immunofluorescence assay.Results:Cell viability was time dependently decreased by exposed to AGEs-BSA (P＜0.01), AGEs-BSA can time dependently increase NO, iNOS, ET-1 concentration and up regulate ET-1 mRNA expression, decrease eNOS secretion and down regulate eNOS mRNA expression (P<0.05), and elevate the fluorescence intensity of p38MAPK in nucelus. While cell viability, secretion of NO、iNOS、ET-1, mRNA expression of ET-1 was significantly decreased after incubation with EMs compared to that with AGEs-BSA (P<0.05), secretion and mRNA expression of eNOS had opposite changes (P<0.05), which in a time and concentration dependent manner. EMs also decreaed the fluorescence intensity of p38MAPK in nucelus compared to AGEs-BSA group.Conclusion:It was demonstrated that AGEs-BSA decreased the cell viability of HUVECs, and impaired the function of secreting endothelial active factors such as NO、eNOS、iNOS、ET-1. EMs has a certain protection effect on AGEs-BSA-induced injury in HUVECs as time and concentration dependent manner, and a inhibitory effect on the expression of p38MAPK in the nucelus activated by AGEs-BSA.更多还原