一氧化氮合酶及其调节

一氧化氮合酶（ＮＯＳ）包括内皮细胞性、神经原性和诱生性三种，其基因分别位于人第７、１２和１７号染色体上。ＮＯＳ是高度保守性蛋白，种属间同一ＮＯＳ的氨基酸同源性大于８０％，但不同ＮＯＳ间仅５０％左右。业已证明ＮＯＳ是非均一性酶，同一酶可存在于不同细胞，而一种细胞可有一种以上的酶。ＮＯＳ的活性受基因转录、翻译、翻译后修饰等多环节调控。对ＮＯＳ的调控研究可指导ＮＯ介导的疾病进行针对性防治     

低氧抑制猪肺动脉平滑肌细胞MMP-2和MMP-9的表达

目的探讨低氧对猪肺动脉平滑肌细胞（PASMC）分泌基质金属蛋白酶（MMPs）的影响。方法采用酶谱法测定PASMC培养基中MMP-2和MMP-9的酶活性，免疫印迹法检测培养基中MMP-2和MMP-9的蛋白表达，免疫组化法测定细胞原位MMP-2和MMP-9的蛋白表达，RT-PCR法检测mRNA的表达。结果低氧后PASMC分泌的MMP-2酶活性、细胞内外蛋白表达量、mRNA表达量均下降；MMP-9酶活性、细胞外蛋白表达量下降，而细胞内蛋白表达无变化。结论低氧可抑制PASMC分泌MMP-2和MMP-9的酶活性，其机制可能是低氧影响PASMC中MMP-2基因的转录、影响MMP-9蛋白表达后的分泌与活化，导致MMP-2和MMP-9酶活性的改变。     

人肺动脉平滑肌细胞中微小RNA-210通过MKP-1负性调节低氧下细胞的增殖

 目的:研究低氧条件下人肺动脉平滑肌细胞中微小RNA-210(miR-210)与丝裂原活化蛋白激酶磷酸酶1 (MKP-1)的关系,是否通过MKP-1调节肺动脉平滑肌细胞增殖及其机制。方法:选用4~8代的人肺动脉平滑肌细胞,共分为12组,其中21% O2正常氧及1% O2低氧培养箱各6组,分别给予转染miR-210抑制剂、增强剂、MKP-1 siRNA等处理,提取RNA和miRNA,并采用实时定量PCR法检测各组平滑肌细胞中miR-210和MKP-1 mRNA的表达,提取蛋白,并用Western blotting法比较各组MKP-1蛋白水平的表达,MTT法检测肺动脉平滑肌细胞的增殖情况。结果:低氧下,人肺动脉平滑肌细胞中miR-210及MKP-1的表达均明显增加,抑制miR-210表达使MKP-1的表达增加并可抑制低氧诱导的细胞增殖,miR-210的过表达可抑制低氧诱导的MKP-1表达上调,但不影响细胞增殖,MKP-1基因沉默后,低氧下miR-210抑制剂对细胞增殖的抑制作用消失。结论: 低氧下的人肺动脉平滑肌细胞中,MKP-1是miR-210的一个新的靶基因,MKP-1可以介导miR-210抑制剂对人肺动脉平滑肌细胞增殖的负性调节作用。     

LPS和几种促炎细胞因子促进人血管平滑肌细胞iNOS mRNA的表达

ＬＰＳ、ｒｈｌＬ－１β、ｒｈＩＬ－６、ｒｈＴｈＦ－ａ和ｒｈＩＦＮ－γ单独或两两组合加于培养的人血管平滑肌细胞（ｈＶＳＭＣ）,测定了ｈＶＳＭＣＮＯＳ活性,ｃＧＭＰ含量,ｉＮＯＳｍＲＮＡ表达丰度和培养液中ＮＯ２浓度。ＬＰＳ和细胞因子都能诱导ｈＶＳＭＣ表达ｉＮＯＳ　ｍＲＮＡ,升高ｈＶＳＭＣ　ＮＯＳ活性、ｃＧＭＰ含量和培养液中ＮＯ２浓度;ＴＮＦ－ａ是受试细胞因子中唯一能与另外三种细胞因子分别组合都显示出协同效应的细胞因子;ＬＰＳ和其余三种细胞因子间的两两组合都没有显示出协同效应;本研究结果表明：ＬＰＳ、ｒｈＴＮＦ－α、ｒｈＩＬ－１β、ｒｈＩＬ－６、ｔｈＴｈＦ－α、ｔｈＩＦＮ－γ都能诱导ｈＶＳＭＣ合成ＮＯ;ＴＮＦ－α可能是诱导ｈＶＳＭＣｉＮＯＳ表达的致炎细胞因子中最为重要的。     

缺氧性肺动脉高压时一氧化氮合酶、左旋精氨酸及亚硝基左旋精氨酸甲酯的研究

目的研究一氧化氮（ＮＯ）在缺氧性肺动脉高压（ＨＰＨ）发病中的作用。方法用烟酰胺腺嘌呤二核苷酸磷酸—黄递酶（ＮｉｃｏｔｉｎａｍｉｄｅＡｄｅｎｉｎｅＤｉｎｕｃｌｅｏｔｉｄｅＰｈｏｓｐｈａｔｅＤｉａｐｈｏｒａｓｅ，ＮＡＤＰＨｄ）和免疫组化ＡＢＣ方法检测原生型和诱生型一氧化氮合酶（ｃＮＯＳ、ｉＮＯＳ）在正常和缺氧大鼠肺内的表达和分布，同时观察左旋精氨酸（Ｌａｒｇ）和亚硝基左旋精氨甲酯（ＬＮＡＭＥ）对正常和缺氧大鼠肺循环的影响。结果ＨＰＨ组，ＮＡＤＰＨｄ阳性反应见于大血管内皮、平滑肌、支气管粘膜上皮及小血管内皮和平滑肌中，而后者在正常时未见阳性反应；ｃＮＯＳ在肺血管内皮和支气管粘膜上皮中的表达明显减弱，甚至消失，而正常时不表达ｉＮＯＳ的肺血管内皮和血管、支气管平滑肌在ＨＰＨ组出现了阳性表达；缺氧时补充Ｌａｒｇ和ＬＮＡＭＥ，与单纯缺氧相比，对右心室肥大和肺血管重建无影响。结论缺氧时ｃＮＯＳ被抑制，可能对ＨＰＨ的形成具有一定的作用；而ｉＮＯＳ的诱导表达，则可能对ＨＰＨ的形成具有阻止作用。     

人肺动脉平滑肌细胞中精氨酸酶Ⅱ与微小RNA-17的相互作用CSCD

背景:已有研究证实microRNA-17-92簇在肺动脉高压中发挥着重要作用,精氨酸酶Ⅱ又参与低氧诱导的肺动脉平滑肌细胞的增殖,而人肺动脉平滑肌细胞中微小RNA-17与精氨酸酶Ⅱ之间的相互作用尚未见研究报道。目的:分析人肺动脉平滑肌细胞中,微小RNA-17与精氨酸酶Ⅱ的关系及其相互作用机制。方法:选用4-8代的人肺动脉平滑肌细胞,分别给予转染微小RNA-17的抑制剂、增强剂、精氨酸酶Ⅱ小干扰RNA等处理,分别在常氧(体积分数21%O2)及低氧(体积分数1%O2)条件下培养。提取RNA和微小RNA,采用实时定量PCR法检测各组平滑肌细胞中微小RNA-17和精氨酸酶ⅡmRNA的表达,提取蛋白,采用Western blot法比较各组细胞中精氨酸酶Ⅱ等蛋白水平的表达。结果与结论:低氧下人肺动脉平滑肌细胞中微小RNA-17及精氨酸酶Ⅱ的表达均明显增加,抑制微小RNA-17的表达可阻止低氧诱导的精氨酸酶Ⅱ的表达增加,微小RNA-17的过表达可使精氨酸酶Ⅱ表达上调,精氨酸酶Ⅱ基因敲除后,低氧诱导的微小RNA-17的表达受到抑制。提示人肺动脉平滑肌细胞中,精氨酸酶Ⅱ是微小RNA-17的一个新的靶基因,而且精氨酸酶Ⅱ可以反馈调节微小RNA-17的表达。     

人肺动脉平滑肌细胞中精氨酸酶Ⅱ与微小RNA-17的相互作用

背景:已有研究证实microRNA-17-92簇在肺动脉高压中发挥着重要作用,精氨酸酶Ⅱ又参与低氧诱导的肺动脉平滑肌细胞的增殖,而人肺动脉平滑肌细胞中微小RNA-17与精氨酸酶Ⅱ之间的相互作用尚未见研究报道。目的:分析人肺动脉平滑肌细胞中,微小RNA-17与精氨酸酶Ⅱ的关系及其相互作用机制。方法:选用4-8代的人肺动脉平滑肌细胞,分别给予转染微小RNA-17的抑制剂、增强剂、精氨酸酶Ⅱ小干扰RNA等处理,分别在常氧(体积分数21%O2)及低氧(体积分数1%O2)条件下培养。提取RNA和微小RNA,采用实时定量PCR法检测各组平滑肌细胞中微小RNA-17和精氨酸酶ⅡmRNA的表达,提取蛋白,采用Western blot法比较各组细胞中精氨酸酶Ⅱ等蛋白水平的表达。结果与结论:低氧下人肺动脉平滑肌细胞中微小RNA-17及精氨酸酶Ⅱ的表达均明显增加,抑制微小RNA-17的表达可阻止低氧诱导的精氨酸酶Ⅱ的表达增加,微小RNA-17的过表达可使精氨酸酶Ⅱ表达上调,精氨酸酶Ⅱ基因敲除后,低氧诱导的微小RNA-17的表达受到抑制。提示人肺动脉平滑肌细胞中,精氨酸酶Ⅱ是微小RNA-17的一个新的靶基因,而且精氨酸酶Ⅱ可以反馈调节微小RNA-17的表达。     

低氧对大鼠肺动脉平滑肌细胞中细胞因子信号转导负调控因子3基因表达的影响

目的：研究低氧对大鼠肺动脉平滑肌细胞（PASMC）中细胞因子信号转导负调控因子3（SOCS3）基因表达的影响。方法： 低氧处理体外培养的大鼠肺动脉平滑肌细胞，采用半定量RT-PCR，免疫细胞化学法和Western blotting法检测常氧组，低氧2 h、6 h、10 h、16 h、24 h组SOCS3 mRNA和蛋白表达水平。结果：常氧培养肺动脉平滑肌细胞SOCS3 mRNA表达呈阴性，低氧培养2 h组肺动脉平滑肌细胞中SOCS3 mRNA表达升高，6 h组达高峰，10 h组开始下降，24 h检测不到其表达；免疫细胞化学定位分析示SOCS3蛋白仅于肺动脉平滑肌细胞胞浆内表达；Western blot定量分析示SOCS3蛋白与SOCS3 mRNA表达的改变基本一致。 结论： 低氧刺激肺动脉平滑肌细胞中SOCS3表达，提示SOCS3在低氧所致肺血管重建过程中可能发挥重要调节作用。     

一氧化氮合酶在胃肠道的分布规律及意义

一氧化氮合酶（ＮＯＳ）是合成一氧化氮（ＮＯ）的关键酶，它存在于体内多种细胞中，可分为ｃＮＯＳ和ｉＮＯＳ两大类，研究表明，ｃＮＯＳ广泛分布于胃肠道，其活性及分布异常可能与某些胃肠疾病的发生、发展有关。     

抗氧化剂和NFκB对大鼠肺微血管内皮细胞iNOS表达的影响

目的：探讨ＮＦкＢ在大鼠肺微血管内皮细胞ｉＮＯＳ基因诱导表达中的作用及抗氧化睦内皮细胞ｉＮＯＳ诱导表达的影响及其机制。方法：Ｇｒｉｅｓｓ法测定细胞上清液ＮＯ２水平以反映ＮＯ的生成,Ｎｒｔｈｅｒｎ印迹分析ｉＮＯＳｍＲＮＡ水平,ＥＭＳＡ法测定细胞核内ＮＦкＢ的结合活性。结果：抗氧化可阻断内皮细胞培养体系ＬＰＳ和ＴＮＦα诱导的ＮＯ生成及ｉＮＯＳｍＲＮＡ的表达;ＬＰＳ和ＴＮＦα可诱导大鼠肺微血管内皮细胞Ｎ     

缺氧性肺动脉高压时一氧化氮合酶,左旋精氨酸及亚硝基左旋？…

研究一氧化氮在缺氧肺动脉高压发病中的作用。方法烟酰胺腺嘌呤二核苷酸磷酸－黄递酶和免疫组化ＡＢＣ方法检测原生型和诱型一氧化氮合酶在正常和缺氧在鼠肺内的表达和分布。     

缺氧诱导因子-1在缺氧诱导一氧化氮合成酶基因表达中的作用

目的 研究缺氧诱导因子-1(HIF-1)在一氧化氮合成酶基因缺氧诱导反应中的作用。方法 体外合成具有HIF-1特异结合位点的DNA片段(红细胞生成素3'-增强子片段),借助脂质体,转入培养的鼠主动脉内皮细胞和肺微血管细胞,用半定量RT-PCR方法测定诱导型一氧化氮合成酶(iNOS)mRNA。结果 (1)大鼠主动脉内皮细胞、肺微血管内皮细胞在常氧下培养,有iNOS基因表达;(2)缺氧能诱导这两种细胞iNOS基因表达增加;(3)野生型EPO3'-增强子片段能阻断缺氧对内皮细胞iNOS基因表达的诱导作用,而突变片段则无此作用。结论 在iNOS基因序列中,可能存在EPO3'-端增强子片段,其参与内皮细胞的缺氧反应。     

Autocrine function of inducible nitric oxide synthase and cyclooxygenase-2 in proliferation of human and rat pulmonary artery smooth-muscle cells: species variation.

Pulmonary hypertension is characterized by hypertrophy and hyperplasia of vascular smooth muscle occurring via an unknown mechanism. Cyclooxygenase (COX)-2 and inducible nitric oxide synthase (iNOS) are expressed under inflammatory conditions and produce mediators that regulate growth in some tissues. We have therefore addressed the question of COX-2 and iNOS involvement in proliferation of human and rat pulmonary artery (PA) smooth-muscle cells (SMC). Interleukin (IL)-1beta suppressed proliferation of both human and rat PA SMC. Moreover, IL-1beta induced COX-2 expression in both cell types. By contrast, IL-1beta stimulated the expression of iNOS protein in rat cells only. COX-2 induced in human cells inhibited proliferation, whereas COX-2 products in rat cells were without affect. However, iNOS activity in rat cells suppressed their proliferation. We conclude that human and rat evolution has diverged such that COX-2 and iNOS, although induced by the same mediator, have different levels of activity and functions in the two species. In humans, induction of COX-2 during pulmonary hypertension may be beneficial for long-term treatment of this disease.     

低氧对人脐静脉内皮细胞一氧化氮和内皮素-1的生成及诱导型一氧化氮合酶mRNA表达的影响

目的：通过观察低氧对人脐静脉内皮细胞(HUVECs)分泌一氧化氮(NO）、内皮素-1(ET-1)以及诱导型一氧化氮合酶(iNOS)mRNA 表达的影响，探讨低氧性肺动脉高压(HPH)形成的机制。 方法： 在离体培养人脐静脉内皮细胞基础上，采用硝酸还原酶和放射免疫分析方法检测了低氧（3％O2）培养6 h、12 h和24 h人脐静脉内皮细胞分泌NO和ET-1的变化，同时运用半定量RT-PCR对iNOS mRNA表达情况进行分析。 结果： 低氧组各时点培养液中NO2-/NO3-和ET-1水平显著高于常氧组(P<0.01)，同时观察到iNOS基因mRNA的表达也相应增高(P<0.01)。 结论： 低氧能刺激人脐静脉内皮细胞生成释放NO和ET-1, NO生成增多与低氧正调iNOS基因mRNA的表达有关。     

一氧化氮与脓毒性休克的关系

在脓毒性休克中,巨噬细胞和血管平滑肌细胞等细胞的诱导型一氧化氮合酶(inducible nitric oxide synthase,iNOS)表达上调,使血管内皮源性舒张因子即一氧化氮(nitric oxide,NO)生成增多,引起血管过度扩张、微循环淤血、血管平滑肌对缩血管物质的反应性降低、心肌功能抑制等,导致严重的...     

慢性低O2高CO2肺动脉高压大鼠血浆NO和肺动脉NOS及其基因表达的变化

目的：探讨一氧化氮体系在慢性低O₂高CO₂肺动脉高压形成中的作用。方法：雄性Sprague-Dawley大鼠分为正常对照组和4周低O₂高CO₂肺动脉高压组。测定血浆NO含量,免疫组化法检测肺细小动脉cNOS和iNOS活性,原位杂交法检测其cNOS mRNA和iNOS mRNA的表达。结果：肺动脉高压组血浆NO含量、肺细小动脉cNOS活性和cNOS mRNA表达显著低于对照组（均P<0.01）,而iNOS活性和iNOS mRNA表达明显高于对照组（均P<0.01）。结论：低O₂高CO₂时肺动脉NOS活性和NOS mRNA表达的改变引起的NO变化参与了肺动脉高压的形成。     

Hypoxic induction of cox-2 regulates proliferation of human pulmonary artery smooth muscle cells

Chronic hypoxia-induced pulmonary hypertension results partly from proliferation of smooth muscle cells in small peripheral pulmonary arteries. Therefore, we examined the effect of hypoxia on growth of pulmonary artery smooth muscle cells (PASMCs) from human distal pulmonary arteries. Initial studies identified that serum-induced proliferation of explant-derived PASMCs was inhibited under hypoxic conditions (3-4 kPa in medium). However, selection of hypoxia-stimulated cells was achieved by culturing cells at low density under conditions of prolonged hypoxia (1-2 wk). In hypoxia-inhibited and -stimulated cells, Western blotting revealed hypoxic induction of cyclooxygenase (COX)-2, which was dependent on the activation of p38(MAPK), but not COX-1, inducible nitric oxide synthase (iNOS), or hemoxygenase-1 (HO-1). Hypoxic induction of COX-2 was also observed in the media of pulmonary arteries in lung organ culture. Hypoxia induced a 4- to 5-fold increase (P < 0.001) in prostaglandin (PG)E(2), PGD(2), PGF(2alpha), and 6-keto-PGF(1alpha) release from PASMCs. Hypoxic inhibition of proliferation was attenuated by incubation with indomethacin (10 micro M), or the COX-2 antagonist, NS398 (10 micro M), but not by the COX-1 antagonist, valeryl salicylate (0.5 mM). In conclusion, we have isolated cells from human peripheral pulmonary arteries that are either inhibited or stimulated by culture under hypoxic conditions. In both cell types hypoxia modulates cell proliferation by induction of COX-2 and production of antiproliferative prostaglandins. Induction of COX-2 may contribute to the inhibition of hypoxia-induced pulmonary vascular remodeling.     

Nitric oxide-mediated mechanism of neuronal nitric oxide synthase and inducible nitric oxide synthase expression during hypoxia in the cerebral cortex of newborn piglets

Previously, we have shown that hypoxia results in increased generation of nitric oxide free radicals in the cerebral cortex of newborn piglets that may be due to up-regulation of nitric oxide synthases, neuronal nitric oxide synthase and inducible nitric oxide synthase. The present study tests the hypothesis that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase in the cerebral cortex of newborn piglets and that the increased expression is nitric oxide-mediated. Newborn piglets, 2-4 days old, were divided to normoxic (n=4), hypoxic (n=4) and hypoxic-treated with 7-nitro-indazole-sodium salt, a selective neuronal nitric oxide synthase inhibitor (hypoxic-7-nitro-indazole-sodium salt, n=6, 1 mg/kg, 60 min prior to hypoxia). Piglets were anesthetized, ventilated and exposed to an FiO2 of 0.21 or 0.07 for 60 min. Cerebral tissue hypoxia was documented biochemically by determining ATP and phosphocreatine. The expression of neuronal nitric oxide synthase and inducible nitric oxide synthase was determined by Western blot using specific antibodies for neuronal nitric oxide synthase and inducible nitric oxide synthase. Protein bands were detected by enhanced chemiluminescence, analyzed by imaging densitometry and the protein band density expressed as absorbance (OD x mm(2)). The density of neuronal nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 41.56+/-4.27 in normoxic, 61.82+/-3.57 in hypoxic (P<0.05) and 47.80+/-1.56 in hypoxic-7-nitro-indazole-sodium salt groups (P=NS vs normoxic), respectively. Similarly, the density of inducible nitric oxide synthase in the normoxic, hypoxic and hypoxic-7-nitro-indazole-sodium salt groups was: 105.21+/-9.09, 157.71+/-13.33 (P<0.05 vx normoxic), 117.84+/-10.32 (p=NS vx normoxic), respectively. The data show that hypoxia results in increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase proteins in the cerebral cortex of newborn piglets and that the hypoxia-induced increased expression is prevented by the administration of 7-nitro-indazole-sodium salt. Furthermore, the neuronal nitric oxide synthase inhibition prevented the inducible nitric oxide synthase expression for a period of 7 days after hypoxia. Since administration of 7-nitro-indazole-sodium salt prevents nitric oxide generation by inhibiting neuronal nitric oxide synthase, we conclude that the hypoxia-induced increased expression of neuronal nitric oxide synthase and inducible nitric oxide synthase is mediated by neuronal nitric oxide synthase derived nitric oxide. We speculate that during hypoxia nitric oxide-mediated up-regulation of nitric oxide synthases will continue the perpetual cycle of nitric oxide generation-->NOS up-regulation-->nitric oxide generation resulting in hypoxic neuronal death.     

Interplay of endothelial and inducible nitric oxide synthases modulates the vascular response to ischaemia-reperfusion in the rabbit lung

Aim: Lung ischaemia–reperfusion induces nitric oxide synthesis and reactive nitrogen species, decreasing nitric oxide bioavailability. We hypothesized that in the ventilated lung, this process begins during ischaemia and intensifies with reperfusion, contributing to ischaemia–reperfusion‐induced pulmonary vasoconstriction. The aim was to determine whether ischaemia–reperfusion alters inducible and endothelial nitric oxide synthase expression/activity, reactive nitrogen species generation, and nitric oxide bioavailability, potentially affecting pulmonary perfusion. Methods: Ischaemia–reperfusion was induced for various times in anesthetized rabbits with ventilated lungs by reversibly occluding the right pulmonary artery and initiating reperfusion. Nitric oxide synthase activity/expression and phosphorylation, reactive nitrogen species generation and total nitrate/nitrite were determined in lung tissue. Results: Inducible nitric oxide synthase expression and activity, and reactive nitrogen species formation coincided with increased pulmonary vascular resistance during reperfusion and increased with ischaemia duration, further increasing after 2‐h reperfusion. Total nitrate/nitrite also increased with ischaemia but decreased after 2‐h reperfusion. Pre‐treatment with an inducible nitric oxide synthase inhibitor (1400W; Cayman Chemical Company, Ann Arbor, MI, USA) attenuated inducible nitric oxide synthase activity, reactive nitrogen species generation and pulmonary vascular resistance, but did not affect total nitrate/nitrite. Endothelial nitric oxide synthase expression was unchanged by ischaemia–reperfusion; however, its phosphorylation on serine 1177 and dephosphorylation on threonine 495 was uncoupled, suggesting decreased endothelial nitric oxide synthase activity. 1400W prevented uncoupling of endothelial nitric oxide synthase phosphorylation, maintaining its activity during reperfusion. Conclusion: Ischaemia–reperfusion up‐regulates inducible nitric oxide synthesis and/activity, which coincides with reduced endothelial nitric oxide synthase activity as suggested by its uncoupling and may contribute to ischaemia–reperfusion‐induced pulmonary vasoconstriction.