精氨酸升压素对大鼠心肌成纤维细胞诱导型一氧化氮合酶-一氧化氮系统活性的影响

目的　研究精氨酸升压素 (AVP)对大鼠心肌成纤维细胞诱导型一氧化氮合酶 (iNOS) 一氧化氮 (NO)系统活性的影响。方法　胰酶消化法分离培养Sprague Dawley仔鼠心肌成纤维细胞 ,采用硝酸还原酶法、蛋白质印迹和逆转录 聚合酶链式反应观察AVP对心肌成纤维细胞的NO含量、iNOS蛋白水平和iNOSmRNA表达的影响。结果　 (1)不同浓度AVP干预下 ,心肌成纤维细胞的NO含量、iNOS蛋白水平和iNOSmRNA表达都随AVP浓度的增高而增加。其中 10 -7mol/LAVP组和 10 -6mol/LAVP组的NO含量 [(6 9 0 5± 5 5 6 ) μmol/L和 (6 2 86± 6 0 5 ) μmol/L]、iNOS蛋白水平 (0 73± 0 0 6和 0 6 4± 0 0 5 )和iNOSmRNA表达 (0 70± 0 0 3和 0 6 6± 0 0 6 )都显著高于对照组 [(2 9 34± 5 34)μmol/L ,0 2 0± 0 0 5 ,0 2 5± 0 0 4 ]、10 -9mol/LAVP组 [(31 79± 6 5 9) μmol/L ,0 2 4± 0 0 7,0 30±0 0 6 ]和 10 -8mol/LAVP组 [(36 87± 7 89) μmol/L ,0 30± 0 0 9,0 31± 0 0 3]。但 10 -6mol/LAVP组的NO含量、iNOS蛋白水平和iNOSmRNA表达都低于 10 -7mol/LAVP组。 (2 ) 10 -7mol/LAVP干预下CFs的NO含量、iNOS蛋白水平和iNOSmRNA表达都随培养时间的延长而增加。其中 2 4h组和 36h组的NO含量 [(6 5     

胰岛素对牛胸主动脉内皮细胞血管内皮生长因子表达的影响及其与一氧化氮合酶的关系

目的:评价胰岛素对内皮细胞血管内皮生长因子(VEGF)表达的影响及其与一氧化氮合酶(NOS)的关系。方法:取新生的小牛胸主动脉,作血管内皮细胞原代及传代培养,取4~6代培养细胞用于实验;应用不同浓度的胰岛素(30、300、3000mU/L)和NOS抑制剂——L-NAME干预培养过程,48h后取内皮细胞应用免疫组化法测定VEGF和一氧化氮合酶3(NOS3)的表达水平。结果:低浓度胰岛素(30、300mU/L)组内皮细胞VEGF和NOS3表达明显高于高浓度(3000mU/L)组(均P<0.01);胰岛素加L-NAME组VEGF和NOS3表达明显低于单纯胰岛素组(均P<0.05)。结论:低浓度胰岛素促进内皮细胞VEGF表达,高浓度胰岛素可抑制内皮细胞VEGF表达,其机制可能是不同浓度胰岛素促进或抑制NOS3的活性。     

四氢生物喋呤对内皮细胞产生一氧化氮和超氧阴离子的影响

目的探讨四氢生物喋呤(BH4)对人脐静脉内皮细胞产生一氧化氮(NO)和超氧阴离子(O-2)的影响.方法在培养液中分别加入不同浓度的D-葡萄糖、胰岛素和BH4,24 h后取细胞培养液分别测定一氧化氮合酶(NOS)、超氧化物歧化酶(SOD)活性、NO和O-2浓度.结果BH4(10、100、500μmol/L)使内皮细胞NOS活性增高,500 μmol/L BH4使内皮细胞NO产生增加,10或100 μmol/LBH4对内皮细胞产生NO有增加的趋势,但与对照组比较无显著性差异(P＞0.05);25 mmol/L葡萄糖+BH4(10、100、500 μmol/L)对内皮细胞产生NO与对照组比较无显著性差异(P＞0.05);高浓度胰岛素(10、100、1 000 mU/L)+BH 4(10、100、500 μmol/L)使内皮细胞NOS活性增强,NO产生增加.BH4(10、100、500μmol/L)对内皮细胞SOD活性无明显影响,但可以改善25 mmol/L葡萄糖对内皮细胞SOD活性的影响;胰岛素+BH4对内皮细胞SOD活性无明显影响(P＞0.05).BH4(10、100、500 μmol/L)使内皮细胞产生O-2减少,并可以改善25 mmol/L葡萄糖对内皮细胞产生O-2影响;胰岛素+BH4组O-2浓度明显低于对照组和不同浓度胰岛素组(P＜0.01).结论BH4可以增加培养的人脐静脉内皮细胞NOS活性,使NO产生增加而使O-2水平下降.     

胰岛素对牛胸主动脉内皮细胞血管内皮生长因子受体表达的影响及其与一氧化氮合酶活性的关系

目的　评价胰岛素对培养的牛胸主动脉内皮细胞血管内皮生长因子 (VEGF)受体flt 1、flk 1 KDR表达的影响及其与一氧化氮合酶 (NOS)的关系。方法　取新生的小牛胸主动脉 ,作血管内皮细胞原代及传代培养 ,取 4 6代培养细胞用于实验 ;应用不同浓度 ( 30mU L、30 0mU L、30 0 0mU L)的胰岛素和NOS抑制剂 (L NAME)干预培养过程 ,4 8h后取培养细胞 ,应用免疫组化法测定flt 1、flk 1 KDR和NOS3的表达水平。结果　不同浓度胰岛素孵育组后内皮细胞flt 1、flk 1 KDR的表达水平差异无显著性 ,L NAME孵育后各组内皮细胞flt 1、flk 1 KDR的表达水平较单纯胰岛素孵育组差异无显著性。结论　胰岛素对内皮细胞flt 1、flk 1 KDR的表达无直接影响 ;内皮细胞NOS3的活性不是内皮细胞VEGF受体flt 1、flk 1 KDR表达的主要影响因素     

涎腺腺癌组织中VEGF和NOS的表达及意义

血管内皮生长因子(VEGF)是血管生成过程中的关键因子，其过度表达与肿瘤生长、侵袭及转移关系密切。研究发现，催化一氧化氮(NO)生成的关键酶一氧化氮合成酶(NOS)活性的增高与肿瘤血管生成关系密切。笔者检测了涎腺腺癌VEGF、诱导型NOS(iNOS)和内皮型NOS(eNOS)的表达，以观察VEGF、iNOS和eNOS在涎腺腺癌中的表达及其相关性，探讨NO和VEGF的相互作用及其在促肿瘤生长中的作用机制。     

尾加压素Ⅱ对大鼠胸主动脉及肠系膜动脉各部分产生一氧化氮的影响

目的取离体大鼠胸主动脉和肠系膜动脉,将UⅡ与血管各层(内、中、外膜)组织共同孵育,观察UⅡ对NOS活性及NO生成的影响,以探讨UⅡ的血管活性效应的机理.材料与方法分离雄性SD大鼠胸主动脉和肠系膜动脉,将胸主动脉各层分离,上述组织分别加入不同浓度尾加压素Ⅱ在37 ℃、通以95%O2～5%CO2气体条件下进行血管组织孵育,孵育时间为2小时和4小时.测定孵育液中亚硝酸盐含量及组织中一氧化氮合酶活性.取最适浓度及最佳时间点,孵育血管后进行诱导型一氧化氮合酶免疫组织化学染色进行表达定位.结果尾加压素Ⅱ(10-9、10-8 mol/L)可以引起胸主动脉外膜一氧化氮生成增加,一氧化氮合酶活性增强,P<0.05,免疫组化证实血管外膜诱导型一氧化氮合酶表达呈强阳性,孵育2小时和4小时没有显著性差异,P>0.05;10-10～10-8 mol/L浓度尾加压素Ⅱ可以浓度依赖性、时间依赖性刺激肠系膜动脉一氧化氮生成,免疫组化证实诱导型一氧化氮合酶在血管外膜和中膜表达增加,但NOS活性没有明显变化.结论 UⅡ可以刺激胸主动脉和肠系膜动脉血管外膜产生NO.UⅡ对胸主动脉及肠系膜动脉NO/NOS系统影响不同,可能是UⅡ作用于血管引起不同生物学效应的机制之一.     

四氢生物喋呤对内皮细胞产生一氧化氮和超氧阴离子的影响

目的 探讨四氢生物喋呤(BH_4)对人脐静脉内皮细胞产生一氧化氮(NO)和超氧阴离子(O_2~(?))的影响。方法 在培养液中分别加入不同浓度的D—葡萄糖、胰岛素和BH_4，24h后取细胞培养液分别测定一氧化氮合酶(NOS)、超氧化物歧化酶(SOD)活性、NO和O_2~(?)浓度。结果 BH_4(10、100、500μmol／L)使内皮细胞NOS活性增高，500μmol／L BH_4使内皮细胞NO产生增加，10或100μmol／L BH_4对内皮细胞产生NO有增加的趋势，但与对照组比较无显著性差异(P＞0.05)；25mmol／L葡萄糖 BH_4(10、100、500μmol／L)对内皮细胞产生NO与对照组比较无显著性差异(P＞0.05)；高浓度胰岛素(10、100、1000mU／L) BH_4(10、100、500μmol／L)使内皮细胞NOS活性增强，NO产生增加。BH_4(10、100、500μmol／L)对内皮细胞SOD活性无明显影响，但可以改善25mmol／L葡萄糖对内皮细胞SOD活性的影响；胰岛素 BH_4对内皮细胞SOD活性无明显影响(P＞0.05)。BH_4(10、100、500μmol／L)使内皮细胞产生O_2~(?)减少，并可以改善25mmol／L葡萄糖对内皮细胞产生O_2~(?)影响；胰岛素 BH_4组O_2~(?)浓度明显低于对照组和不同浓度胰岛素组(P＜0.01)。结论 BH_4可以增加培养的人脐静脉内皮细胞NOS活性，使NO产生增加而使O_2~(?)水平下降。     

尾加压素Ⅱ对大鼠胸主动脉及肠系膜动脉各部分产生一氧化氮的影响

目的　取离体大鼠胸主动脉和肠系膜动脉 ,将UⅡ与血管各层 (内、中、外膜 )组织共同孵育 ,观察UⅡ对NOS活性及NO生成的影响 ,以探讨UⅡ的血管活性效应的机理。材料与方法　分离雄性SD大鼠胸主动脉和肠系膜动脉 ,将胸主动脉各层分离 ,上述组织分别加入不同浓度尾加压素Ⅱ在 37℃、通以 95 %O2 ～ 5 %CO2 气体条件下进行血管组织孵育 ,孵育时间为 2小时和 4小时。测定孵育液中亚硝酸盐含量及组织中一氧化氮合酶活性。取最适浓度及最佳时间点 ,孵育血管后进行诱导型一氧化氮合酶免疫组织化学染色进行表达定位。结果　尾加压素Ⅱ (1 0 - 9、1 0 - 8mol/L)可以引起胸主动脉外膜一氧化氮生成增加 ,一氧化氮合酶活性增强 ,P <0 0 5 ,免疫组化证实血管外膜诱导型一氧化氮合酶表达呈强阳性 ,孵育 2小时和 4小时没有显著性差异 ,P >0 0 5 ;1 0 - 1 0 ～ 1 0 - 8mol/L浓度尾加压素Ⅱ可以浓度依赖性、时间依赖性刺激肠系膜动脉一氧化氮生成 ,免疫组化证实诱导型一氧化氮合酶在血管外膜和中膜表达增加 ,但NOS活性没有明显变化。结论　UⅡ可以刺激胸主动脉和肠系膜动脉血管外膜产生NO。UⅡ对胸主动脉及肠系膜动脉NO/NOS系统影响不同 ,可能是UⅡ作用于血管引起不同生物学效应的机制之一。     

家兔动脉粥样硬化进程中内皮依赖性因子的变化及其相互关系研究

目的　探讨血红素氧合酶-1(HO -1) /一氧化碳(CO)系统与一氧化氮合酶(NOS) /一氧化氮(NO)在动脉粥样硬化中的变化、相互关系及对动脉粥样硬化进程的影响。方法　家兔予以高胆固醇饮食(n=8)以及在高胆固醇饮食的同时经饮水给予L -精氨酸(n=8)或亚硝基左旋精氨酸甲酯(n=8),或经腹腔注射血红素L -赖氨酸盐(n=8)或锌原卟啉9 (ZnPP- IX) (n=8 ),共10周。结果与对照组比较,胆固醇组主动脉NO生成量显著减少,CO生成则明显增加,NOS活性显著降低(P均<0 .01),而HO -1表达升高,主动脉斑块面积达(40. 2±8 .9)% 。与胆固醇组比较,外源性血红素L -赖氨酸盐干预组的主动脉内膜斑块面积[ (26. 6±9 .2)% ]明显缩小,主动脉CO的生成量和HO- 1表达明显升高(P<0. 01),但NOS活性与NO产量较正常对照组显著降低(P<0 .01),与胆固醇组比较则无显著差异;与胆固醇组比较,外源性L 精氨酸显著升高主动脉cNOS活性,增加NO生成量,主动脉斑块面积[ (28. 1±7 .7)% ]明显缩小(P均<0. 01),而HO -1的表达和CO的生成较正常对照组显著升高,与胆固醇组相比差异无统计学意义。与胆固醇组比较,血红素L 赖氨酸盐干预组、L 精氨酸组的主动脉组织内c- myc及c- fos的mRNA和蛋白表达均显著降低,而ZnPP组和亚硝基左旋精氨酸甲酯组则无明显差异。结论　动     

反义MAPK寡核苷酸可抑制血管升压素诱导的心肌成纤维细胞一氧化氮合酶活性

目的探讨反义丝裂素活化蛋白激酶（MAPK）寡核苷酸对血管升压素（VP）诱导的心肌成纤维细胞（CFs）一氧化氮合酶（NOS）活性的抑制作用。方法分离培养SD仔鼠CFs,采用分光光度法和RT-PCR测定CFs NOS活性和诱导型一氧化氮合酶（iNOS）mRNA表达。结果VP显著提高CFs NOS活性和iNOS mRNA表达;反义MAPK寡核苷酸剂量依赖性地抑制VP诱导下CFs NOS活性增高和iNOS mRNA表达增强,其中0.2μmol/L和0.3μmol/L反义MAPK寡核苷酸可将VP诱导下CFs NOS活性和iNOS mRNA表达抑制到与对照组近似水平。结论MAPK参与了VP诱导下CFs NOS活性增高和iNOS mRNA表达增强。     

胰岛素抵抗状态下HepG2细胞纤维蛋白溶解酶原激活物抑？…

目的 观察胰岛素、胰岛素原对ＨｅｐＧ２细胞纤维蛋白溶解酶原激活物抑制物－１（ＰＡＩ－１）基因表达的影响。方法 将ＨｅｐＧ２细胞置于１０＾－７ｍｏｌ／Ｌ胰岛素培液中２４小时使胰素受体下调将ＨｅｐＧ２细胞导成为胰岛素抵抗的ＨｅｐＧ２细胞,然后分别用胰岛素、胰岛素原（１０＾－９ｍｏｌ／Ｌ）持续刺激ＨｅｐＧ２细胞２４小时,检测培养液中ＰＡＩ的活性、含量及胞浆中ＰＡＩ－１ｍＲＮＡ水平。结果（１）基因状态下胰     

Paclitaxel induces thrombomodulin downregulation in human aortic endothelial cells

Patients with paclitaxel-eluting stents are at risk of developing stent thrombosis upon premature discontinuation of dual antiplatelet therapy. In this study, we set out to clarify whether paclitaxel can modulate thrombomodulin expression in human aortic endothelial cells. Human aortic endothelial cells were stimulated with paclitaxel. Methoxyphenyl tetrazolium inner salt cell viability assay, Western blot analysis, real-time polymerase chain reaction, and immunohistochemical assay were performed. In human aortic endothelial cells, paclitaxel (10(-5) to 10(-9) mol/L) treatment for 13 hours caused significant cytotoxicity at drug concentrations greater than 10(-7) mol/L. Paclitaxel (10(-5) to 10(-9) mol/L) treatment for 5 hours downregulated thrombomodulin expression dose-dependently, persisting even at 13 hours. Cotreatment with thrombin and paclitaxel did not alter the effect of paclitaxel on thrombomodulin downregulation. Paclitaxel caused a 0.63-fold decrease in thrombomodulin messenger RNA expression, and thrombin cotreatment did not alter this decrease. In vivo studies confirmed that paclitaxel (10 mg/kg) caused endothelial thrombomodulin downregulation in mice. In summary, paclitaxel downregulates thrombomodulin expression regardless of thrombin stimulation, which is an important factor for patients receiving paclitaxel-eluting stents. Therefore, further designs of drug-eluting stents should consider the influence of the eluted drugs on endothelial thrombogenicity.     

肝细胞一氧化氮合酶的诱导及动力学研究

目的对内毒素和几种细胞因子诱导肝细胞一氧化氮合酶的协同效应及酶动力学参数进行研究.方法原位预灌流和段原酶循环灌流大鼠肝脏、分离肝实质细胞,观察内毒素、IFN-Y、IFN-α、TNFα、IL-lβ、IL-6及不同组合对肝细胞一氧化氮合酶活性、cGMP及NO2-+NO3的影响,分析酶动力学特征及皮质甾与酶诱导的量效关系.结果内毒素+IFN-v+TNFα+IL-lβ(IL-6)组合诱导酶表达效应最显著;酶参数分析显示Km、Vmax分别为108μmol/L和2632pmol@min1mg-1蛋白质,竞争性抑制剂L-NMMA、L-NNA作用的Ki分别为056μmolL及094μmol/L;诱导时间进程显示iNOS活性表达在9h达到峰值,但cGMP及NO2-NO3-的释放持续增加可维持至l8h;地塞米松和氢化可的松抑制肝细胞酶诱导的IC50分别为35×10-8mol/L和26×10-0mol/L.结论肝细胞诱导性一氧化氮合酶的表达依赖特异多细胞因子协同作用,这种可诱导性特征可能在内毒素血症和败血症休克发病机制中具有重要意义.     

胰高血糖素样肽1受体激动剂对高糖诱导的血管内皮细胞NF-κB、ICAM-1和VCAM-1表达的影响

目的观察胰高血糖素样肽1受体激动剂艾塞那肽对高糖诱导的人脐静脉内皮细胞核因子κB(NF-κB)活性及细胞间黏附分子1(ICAM-1)、血管细胞黏附分子1(VCAM-1)表达的影响,探讨胰高血糖素样肽1受体激动剂降糖外对改善糖尿病患者血管内皮损伤的作用及其可能的机制。方法体外培养人脐静脉内皮细胞,以不同浓度艾塞那肽和高糖共同孵育48 h,应用酶联免疫吸附法检测细胞培养上清液中ICAM-1和VCAM-1浓度,应用逆转录聚合酶链反应测定NF-κB p65、ICAM-1和VCAM-1的mRNA表达。结果与正常对照组比较,高糖组NF-κB p65、ICAM-1和VCAM-1的表达显著升高(P<0.01)。艾塞那肽干预后,NF-κB p65、ICAM-1及VCAM-1的表达较高糖组显著降低(P<0.01),并呈剂量依赖性。结论艾塞那肽可能通过抑制高糖环境下NF-κB活性影响其下游黏附分子ICAM-1和VCAM-1的高表达,由此稳定血管内皮环境,减轻高糖引起的内皮细胞损伤,有助于延缓糖尿病动脉粥样硬化病程。     

Expression and relationship between endothelin-1 messenger ribonucleic acid (mRNA) and inducible/endothelial nitric oxide synthase mRNA isoforms from normal and preeclamptic placentas

Preeclampsia is a mainly vascular disease of pregnancy, probably caused by an imbalance between vasodilator and vasoconstrictor agents that results in generalized vasospasm and poor perfusion in many organs. Among these factors, endothelin-1 (ET-1), a potent vasoconstrictor, is highly increased in preeclamptic women, while nitric oxide (NO), a vasodilator of human utero-placental arteries, is reduced in the same patients. The present study was designed to investigate the interactions between ET-1 and the NO system in the feto-placental unit; to this purpose we also examined the messenger ribonucleic acid (mRNA) expression of ET-1, inducible NO synthase (iNOS), and endothelial NOS (eNOS) in human cultured placental trophoblastic cells obtained from preeclamptic (PE) and normotensive (NT) pregnancies. We also studied whether exogenous ET-1 may affect the expression of iNOS and eNOS in human placental trophoblastic cells. Interestingly, by Northern blot analysis we observed an increased ET-1 mRNA expression level in PE trophoblastic cells compared to NT trophoblastic cells. Furthermore, exogenous ET-1 (10(-7) mol/L) was able to up-regulate its own mRNA expression in both NT and PE trophoblastic cells. iNOS and eNOS mRNA expression was then detected, by semiquantitative PCR, in both NT and PE trophoblastic cells. PE trophoblastic cells expressed lower iNOS mRNA levels compared with NT pregnancies. On the contrary, eNOS mRNA expression was higher in PE trophoblastic cells than in NT cells. Moreover, in the presence of ET-1 we observed a decrease in iNOS and an increase in eNOS mRNA expression levels in both NT and PE trophoblastic cells compared with the respective untreated cells. In conclusion, we demonstrate that ET-1 expression is increased in PE cells, whereas iNOS, which represents the main source of NO synthesis, is decreased; conversely, eNOS expression is increased. Finally, ET-1 is able to influence its own as well as NOS isoform expression in normal and PE trophoblastic cultured cells. These findings suggest the existence of a functional relationships between ET(s) and NOS isoforms that could constitute the biological mechanism leading to the reduced placental blood flow and increased resistance to flow in the feto-maternal circulation, which are characteristic of the pathophysiology of preeclampsia.     

不同HO-1表达水平对糖尿病模型大鼠血管舒张功能及NOS的影响

目的 探讨改变血红素加氧酶-1(HO-1)表达水平对糖尿病(DM)大鼠血管舒张功能的影响及与一氧化氮合酶(NOS)/一氧化氮(NO)的关系.方法 以链脲佐菌素(STZ)诱导DM大鼠模型.SD大鼠分成4组:对照组、DM组、正铁血红素(HO-1诱导剂)组、锌原卟啉(HO-1抑制剂)组.应用离体血管张力检测技术观察胸主动脉舒张功能变化;RT-PCR法及比色法分别检测血管组织和血清中诱生型NOS(iNOS)及内皮型NOS(eNOS)的表达和NO含量.结果 与DM组相比,正铁血红素组血管环对乙酰胆碱舒张百分率有所提高,而锌原卟啉组血管舒张反应继续下降.应用正铁血红素可在提高DM大鼠血管和血清eNOS表达的同时降低iNOS/NO表达;而锌原卟啉组血清中iNOS活性及其在血管组织表达均增高.结论 提高HO-1的表达水平有益于改善DM大鼠血管舒张反应失调,这种保护作用与抑制iNOS/NO的生成、上调eNOS表达水平有关.     

Retinoic acid attenuates inducible nitric oxide synthase (NOS2) activation in cultured rat cardiac myocytes and microvascular endothelial cells

S. Grosjean, Y. Devaux, C. Seguin, C. Meistelman, F. Zannad, P.-M. Mertes, R. A. Kelly and D. Ungureanu-Longrois. Retinoic Acid Attenuates Inducible Nitric Oxide Synthase (NOS2) Activation in Cultured Rat Cardiac Myocytes and Microvascular Endothelial Cells. Journal of Molecular and Cellular Cardiology (2001) 33, 933-945. The inducible NO synthase (NOS2) in cardiac tissue contributes to myocardial and coronary inflammation and dysfunction. Several natural (endogenous) hormones such as retinoic acid, the active metabolite of vitamin A, have the ability to attenuate NOS2 activation in inflammatory cells. The aim of this study was to investigate the effect of RA on NOS2 activation in cultured cardiac microvascular endothelial cells (CMEC) and adult rat ventricular myocytes (ARVM). CMEC were stimulated either with a combination of 10 microg/ml lipopolysaccharide (LPS) and 50 IU/ml interferon- gamma (IFN- gamma) or with a combination of 1 ng/ml interleukin-1 beta (IL-1 beta)+IFN- gamma whereas ARVM were stimulated with 1 ng/ml IL-1 beta and 50 IU/ml IFN- gamma in the absence or presence of all-trans retinoic acid (atRA). Activation of the NOS2 pathway was estimated by measurement of mRNA (Northern blot) and protein (Western blot) expression, enzyme activity by conversion of [(3)H]L -arginine to [(3)H]L -citrulline, and nitrite accumulation. NOS2 mRNA half-life was studied in CMEC and ARVM in the presence of actinomycin D. In CMEC and ARVM stimulated with a combination of LPS and/or cytokines, atRA (10(-6), 10(-5)M) significantly (P<0.05) attenuated NOS2 mRNA and protein expression, enzymatic activity and reduced supernatant nitrite concentration. Upon stimulation with LPS/IFN- gamma, atRA significantly decreased NOS2 mRNA half-life. This was not seen after stimulation with IL-1 beta/IFN- gamma. These results document for the first time an effect of RA on NOS2 activation in cardiac cells. They may contribute to the characterization of the immunomodulatory effects of retinoids in myocardial and coronary inflammatory disorders.     

内皮舒张因子在缺氧肺动脉收缩中的作用

本工作在离体灌流肺动脉环模型及培养的牛肺动脉内皮细胞探讨内皮舒张因子 (EDRF/NO)在缺氧肺动脉收缩中的作用。结果发现 ,在内皮完整血管环加入NO合成抑制剂L NNA(10 -4mol/L)、NO合成前体L Arg(10 -2 mol/L)分别升高 ( 80 % ,P <0 .0 1)和降低 (-35 % ,P <0 .0 5 )缺氧肺动脉收缩幅度 ;给予硝酸甘油 (10 -4 mol/L)直接补充NO可降低缺氧肺动脉收缩幅度 ,此效应可被可溶性鸟苷酸环化酶抑制剂亚甲蓝部分逆转 ;用Northern印渍杂交技术显示缺氧使培养的牛肺动脉内皮细胞NO合成酶(NOS)基因表达明显受抑。结果提示缺氧时NOS基因表达受抑及NO合成释放降低可能是缺氧肺动脉收缩的发生机制之一。     

氟致体外培养人脐静脉血管内皮细胞损伤的实验观察

目的 观察不同剂量的氟对体外培养人脐静脉血管内皮细胞(HUVEC)的影响.方法 在HUVEC培养液中加入不同剂量的氟化钠(NaF),分别为0(对照)100、400、700、1000、2000 μmol/L,每组设6个复孔,连续培养48 h,收集细胞培养液与细胞.瑞氏-吉姆萨染色观察细胞形态,吖啶橙荧光染色测定细胞凋亡,四唑氮蓝(MT T)比色法检测细胞活性;分光光度法检测细胞培养液中超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-Px)活性、丙二醛(MDA)水平及诱导型一氧化氮合酶(iNOS)、内皮型一氧化氮合酶(eNOS)活性;RT-PCR法检测细胞iNOS mRNA和eNOS mRNA表达水平;双抗体夹心ELISA法检测细胞培养液中细胞黏附因子(ICAM-1)、血管黏附因子(VCAM-1)水平.结果 随染氟剂量增加,HUVEC细胞数量减少,结构改变;400～2000μmol/L NaF组SOD活性[(6.627±0.213)、(6.668±0.152)、(5.935±0.122)、(4.755±0.182)kU/L]较对照组[(7.457±0.398)kU/L]降低(P＜0.05或＜0.01),GSH-Px活性[(481.284±43.785)、(492.223±16.474)、(382.762±25.167)、(293.687±24.881)kU/L]较对照组[(585.078±47.323)kU/L]降低(P＜0.05或＜0.01),MDA水平[(0.609±0.011)、(0.646±0.016)、(0.852±0.013)、(1.188±0.045)nmol/L]较对照组[(0.512±0.027)nmol/L]升高(P＜0.05或＜0.01);iNOS活性[(3.604±0.115)、(3.615±0.075)、(3.848±0.103)、(4.275±0.079)kU/L]较对照组[(2.798±0.136)kU/L]增强(P均＜0.01),iNOS mRNA表达增强,eNOS活性[(5.539±0.079)、(5.503±0.064)、(5.226±0.142)、(4.809±0.107)kU/L]较对照组[(5.996±0.155)kU/L]减弱(P＜0.05或＜0.01),eNOSmRNA表达减弱;ICAM-1水平[(0.852±0.102)、(0.886±0.061)、(0.961±0.158)、(1.418±0.167)μg/L]较对照组[(0.687±0.046)μg/L]升高(P＜0.05或＜0.01),VCAM-1水平[(2.719±0.197)、(2.946±0.167)、(3.173±0.225)、(3.613±0.153)μg/L]较对照组[(2.375±0.067)μg/L]升高(P均＜0.01).结论 高剂量氟降低抗氧化酶活性,使一氧化氮代谢紊乱,细胞因子异常表达,以此抑制血管内皮细胞生长、结构改变并致细胞凋亡,为高氟致血管内皮损伤的重要因素.

Abstract：

Objective To study the effect of different concentrations of fluoride on cultured human umbilical vein vascular endothelial cells(HUVEC). Methods Different doses of sodium fluoride (NaF) were added to HUVEC culture medium, fluoride concentrations were 0(control), 100,400,700,1000,2000 μmol/L, respectively,6 re-set hole in each group. After continuous culture for 48 h, cells and culture medium were collected. Cell morphology was studied by Wright-Giemsa staining; cells apoptosis was determined by acridine orange fluorescence staining; cell activity was measured by methyl thiazolyl tetrazolium (MTT) assay; superoxide dismutase (SOD),glutathione peroxidase(GSH-Px) activity, malonaldehyde(MDA) content, induced nitricoxide synthase(iNOS), and endothelia nitricoxide synthase(eNOS) activity in cell culture medium were determined by spectrophotometry; cell iNOS mRNA and eNOS mRNA expression were detected by RT-PCR; intercellular adhesion molecule-1 (ICAM-1)and vascular cell adhesion molecule-1 (VCAM-1) levels were detected by double antibody sandwich ELISA method.Results With increased dose of fluoride, HUVEC cells decreased, the structure changed. In 400 - 2000 μmol/L group, the SOD activity[(6.627 ± 0.213), (6.668 ± 0.152), (5.935 ± 0.122), (4.755 ± 0.182)kU/L] was lower than those of the control group[(7.457 ± 0.398)kU/L, P ＜ 0.05 or ＜ 0.01], GSH-Px activity[(481.284 ± 43.785),(492.223 ± 16.474), (382.762 ± 25.167), (293.687 ± 24.881 )kU/L] was also lower than those of the control group [(585.078 ± 47.323)kU/L, P ＜ 0.05 or ＜ 0.01], MDA level[(0.609 ± 0.011 ), (0.646 ± 0.016), (0.852 ± 0.013),(1.188 ± 0.045)nmol/L] was higher than those of the control group[(0.512 ± 0.027)nmol/L, P ＜ 0.05 or ＜ 0.01];iNOS activity[(3.604 ± 0.115), (3.615 ± 0.075), (3.848 ± 0.103), (4.275 ± 0.079)kU/L] also was higher than those of the control group[(2.798 ± 0. 136)kU/L, all P ＜ 0.01], iNOS mRNA expression increased, eNOS activity [(5.539 ± 0.079), (5.503 ± 0.064), (5.226 ± 0.142), (4.809 ± 0. 107)kU/L] decreased compared to those of control group[(5.996 ± 0.155)kU/L, P ＜ 0.05 or ＜ 0.01], eNOS mRNA expression decreased; ICAM-1 levels [(0.852 ± 0. 102), (0.886 ± 0.061 ), (0.961 ± 0.158), (1.418 ± 0. 167)μg/L] increased compared to those of the control group[(0.687 ± 0.046)μg/L, P ＜ 0.05 or ＜ 0.01], VCAM-1 levels[(2.719 ± 0.197), (2.946 ± 0.167),(3.173 ± 0.225 ), (3.613 ± 0. 153 ) μg/L] was higher than those of the control group [(2.375 ± 0.067 ) μg/L, all P ＜0.01]. Conclusions High concentrations of fluoride reduce the activity of antioxidant enzymes, which leads to metabolic disorders of nitric oxide and abnormal cytokines expression, thereby inhibiting vascular endothelial cell growth, structural change and induced apoptosis. This is an important factor in high fluoride-induced vascular endothelial injury.