【摘要】 阐明肿瘤坏死因子α（ＴＮＦα）对内皮细胞一氧化氮（ＮＯ）的产生及两型一氧化氮合酶（ＮＯＳ）ｍＲＮＡ表达的影响及其规律。方法以大鼠肺微血管内皮细胞为材料，测定细胞培养上清液中ＮＯ２－水平并应用定量ＲＴ／ＰＣＲ技术检测内皮细胞ＮＯＳｍＲＮＡ表达水平。结果应用ＴＮＦα（１００ｎｇ／ｍｌ）作用于内皮细胞６ｈ后，发现培养上清液ＮＯ２－水平较对照组显著升高（Ｐ＜０．０５），而６ｈ以内两组无显著差别（Ｐ＞０．０５）。预先应用Ｌ－ＮＭＭＡ、地塞米松、放线菌酮均可阻断ＴＮＦα诱导的ＮＯ２－水平的升高，而应用Ｌ－精氨酸则可进一步升高ＮＯ２－水平。定量ＲＴ－ＰＣＲ结果发现，ＴＮＦα（１００ｎｇ／ｍｌ）作用２４ｈ后，大鼠肺微血管内皮细胞诱导型ＮＯＳ（ｉＮＯＳ）ｍＲＮＡ水平较对照组明显增高，而预先应用地塞米松或放线菌酮后，ｉＮＯＳｍＲＮＡ水平与对照组无明显差异（Ｐ＞０．０５），同时内皮型ＮＯＳ（ｅＮＯＳ）ｍＲＮＡ水平较对照组显著降低，这种下调作用可被放线菌酮阻断，但不被地塞米松阻断。结论①ＴＮＦα可诱导大鼠肺微血管内皮细胞ｉＮＯＳｍＲＮＡ表达而抑制ｅＮＯＳｍＲＮＡ表达，此过程有新合成的蛋白质因子参与；②ＴＮＦα对内皮细胞中两型ＮＯＳ?更多还原

【Abstract】 Purpose To elucidate the regulation of inducible and constitutive nitric oxide synthase gene in endothelial cells by TNFα. Methods The method of rat pulmonary microvascular endothelial cell(RPMEC) culture was improved and the cells were identified with anti endothelial cell antibody CD 31 using ABC method.The concentrations of NO 2 -in the culture media were determined based on Griess reaction.Quantitive RT/PCR technique was performed to quantitate the levels of NOS mRNA in RPMECs.[WT10.HZ〗 Results A marked production of NO 2 -in RPMECs was found after 6 hours’ treatment with TNFα(100ng/ml)( P <0.05).Pretreatment with L-NMMA(500μmol/L),dexamethasone (1μmol/L) or cycloheximide (10μg/ml) significantly decreased NO 2 - production induced by TNFα( P <0.05 vs.control).A further increase was found after adding L-arginine (500μmol/L).The level of iNOS mRNA increased and that of eNOS mRNA decreased significantly after adding TNFα(100ng/ml)to the cell media for 24 h ( P <0.05).Dexamethasone(1μmol/L) blunted the induction of iNOS mRNA.The protein synthesis inhibitor,cycloheximide(10μg/ml),blocked the induction of iNOS mRNA,meanwhile,it also blocked the inhibitory effect of TNFα on eNOS mRNA. Conclusions ① TNFα may induce iNOS gene expression and suppress eNOS gene expression at transcriptional or posttranscriptional level.The upregulation of iNOS and downregulation of eNOS message depend on new protein synthesis.② Different mechanisms are involved in the regulation of TNFα on iNOS and eNOS expression in endothelial cells.③ High concentration of TNFα(100ng/ml) may increase NO production in RPMECs,indicating that NO plays an portant role in the regulation of microvascular movement in physiological and pathological environment.更多还原