辛伐他汀对新生大鼠缺氧缺血后脑皮质区诱导型一氧化氮合酶活性的影响

新生儿缺氧缺血性脑损伤（hypoxic-ischemic braind amage，HIBD）病理过程的发生、发展与多种因素有关，不同亚型的一氧化氮合酶（nitric oxide synthase，NOS）在其中的作用不同，内皮型一氧化氮合酶（endothelial nitric oxide synthase，eNOS）产生的一氧化氮（nitric oxide，N0）有神经保护作用，而神经元型一氧化氮合酶（neuronal nitric oxide synthase，nNOS）与诱导型一氧化氮合酶（inducible nitric oxide synthase，iNOS）产生的NO与神经损伤有关。本研究通过观察新生大鼠缺氧缺血损伤后脑皮质区iNOS活性的变化以及辛伐他汀对其的影响，探讨辛伐他汀对缺氧缺血新生鼠脑的保护作用及可能机制。     

子癎前期患者胎盘内源性一氧化氮合酶抑制物水解酶的表达

子癎前期(pre-eclampsia)是妊娠期特有的疾病,严重威胁母婴安全和健康.但其病因及发病机理还有许多值得探讨之处,近年关注到内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)的抑制物及抑制物水解酶在子癎前期发病中的作用.     

子痫前期-子痫患者胎盘组织中内皮型一氧化氮合酶运输介导物与内皮型一氧化氮合酶的相关性研究

目的:通过测定子痫前期-子痫患者胎盘组织中内皮型一氧化氮合酶运输介导物(endothelial nitric oxide synthase traffic inducer,NOSTR IN)和内皮型一氧化氮合酶(endothelial nitric oxide synthase,eNOS)的表达,探讨它在子痫前期-子痫发病中的作用。方法:采用逆转录-聚合酶链反应(RT-PCR)检测21例子痫前期-子痫患者(病例组)和17例正常晚孕妇女(正常组)胎盘组织中NOSTR IN mRNA的表达;W estern blot检测胎盘组织中NOSTR IN和eNOS蛋白质的表达;分光光度法测定胎盘组织中eNOS的活性;应用硝酸还原酶法测定孕妇静脉血中的NO代谢产物亚硝酸基/亚硝基(NO2-/NO3-)。结果:病例组患者胎盘组织中NOSTR IN mRNA呈强表达,明显高于正常组(P<0.01);W esternblot显示两组胎盘组织中均有58kDa的NOSTR IN蛋白质表达,但病例组表达量明显增高(P<0.01),同时也均有145kDa的eNOS蛋白质表达,两组表达量比较无差异(P>0.05);病例组患者胎盘组织eNOS活性为13.727±3.58 U/mg,与正常组的21.69±3.84U/mg比较降低显著(P<0.01);病例组孕妇外周血清NO2-/NO3-为38.56±8.49μmol/l,与正常组的65.37±9.61μmol/l比较显著降低(P<0.01);病例组胎盘组织中NOSTRN表达水平与eNOS活性呈负相关(r=-0.57,P<0.01)。结论:子痫前期-子痫患者胎盘组织中NOSTR IN表达水平升高,eNOS活性降低,这可能在子痫前期-子痫的发病中起重要作用。     

妊娠期糖尿病孕妇足月妊娠胎盘中一氧化氮合成酶的表达及一氧化氮含量

目的 探讨妊娠期糖尿病(gestational diabetes mellitus,GDM)患者足月妊娠胎盘组织中内皮型一氧化氮合成酶(endothelial nitric oxide synthase,eNOS)、诱导型一氧化氮合成酶(inducible nitric oxide synthase,iNOS)的表达及一氧化氮(nitric oxide,NO)含量变化. 方法 选择2011年10月10日至2012年5月15日在上海交通大学医学院附属第一人民医院行剖宫产分娩的单胎妊娠GDM孕妇为研究对象,分为饮食运动治疗组30例和胰岛素治疗组30例,血糖均控制在正常范围.选择同期分娩的无任何妊娠合并症及并发症的30例孕妇为对照组.采用免疫组织化学染色检测胎盘组织中eNOS和iNOS的表达,酶联免疫吸附试验检测eNOS和iNOS活性以及NO含量.统计学方法采用单因素方差分析、LSD-t检验、非参数秩和检验和非参数两个独立样本的秩和检验. 结果 (1)eNOS在胎盘血管内皮细胞的细胞质和细胞膜中表达.对照组、GDM饮食运动治疗组与GDM胰岛素治疗组3组间的免疫组织化学染色评分[(0.51±0.04)、(0.52±0.04)和(0.50±0.03)分,F=1.248]和活性[(19.42±0.04)、(19.45±0.06)与(19.43±0.07)U/mg prot,F=1.603]比较,差异均无统计学意义(P＞0.05).(2)i NOS在胎盘滋养细胞的细胞质和细胞膜中表达.对照组、GDM饮食运动治疗组与GDM胰岛素治疗组3组间的免疫组织化学染色评分[(0.45±0.23)、(1.16±0.75)与(3.03±0.84)分,H=62.178]和活性[(10.92±1.09)、(13.86±0.40)与(14.38±0.44)U/mg prot,H=54.144]比较,差异均有统计学意义(P＜0.001),GDM组均高于对照组,GDM胰岛素治疗组高于饮食运动治疗组(P＜0.001).(3)对照组、GDM饮食运动治疗组与GDM胰岛素治疗组3组间NO含量[(39.07±10.03)、(80.31±4.81)与(78.53±7.53) μmol/L,H=47.901]比较,差异有统计学意义(P＜0.001),GDM组均高于对照组(P＜0.05),但GDM 2组之间比较,差异无统计学意义(P＞0.001). 结论 在血糖控制理想的GDM患者胎盘组织中,eNOS表达和活性无明显变化,iNOS表达和活性均增加,NO含量的增加与iNOS表达和活性增加可能有关.     

妊高征患者绒毛合体滋养细胞一氧化氮合酶含量及活性相关性的研究

目的　探讨妊高征患者绒毛合体滋养细胞一氧化氮合酶含量与活性的相关性。方法　采用免疫组织化学ABC法和酶组织化学法 (四唑盐法 ) ,检测 32例妊高征患者 (妊高征组 )和 32例正常足月妊娠妇女 (正常组 )绒毛合体滋养细胞一氧化氮合酶 (NOS)含量及活性的改变。结果　两组绒毛均存在eNOS、iNOS抗原及NOS活性的阳性表达 ,且部位相同。两组绒毛血管内皮细胞中eNOS、iNOS抗原及NOS活性的表达强度基本一致 ;而合体滋养细胞中的表达 ,则妊高征组低于正常组。两组合体滋养细胞中 ,NOS活性表达强度的例数分布与eNOS含量表达强度的例数分布呈正相关 ,而与iNOS含量无相关性。中、重度妊高征患者合体滋养细胞中 ,NOS活性与eNOS含量间也呈正相关。结论　绒毛合体滋养细胞eNOS含量及活性的改变 ,可能在妊高征的发病中起重要作用。     

孕鼠不同给氧方式对胎鼠缺氧缺血性脑损伤的作用

围产期缺氧缺血性脑损伤(hypoxic-ischemic brain damage,HIBD)约90%发生在宫内或产时,可导致围产儿死亡或严重后遗症,因此对宫内}tlBD和宫内窘迫进行早期干预具有重要的临床意义.本研究通过建立宫内缺氧缺血胎鼠模型,给孕鼠不同浓度的氧,观察胎鼠脑组织病理学变化,并通过免疫组织化学方法测定脑组织中诱导型一氧化氮合酶(indueible nitric oxide synthase,iNOS)的表达,探讨不同氧浓度对胎儿HIBD的作用.     

T细胞淋巴瘤侵袭转移诱导因子l(Tiam1)在动情周期小鼠子宫内膜中的表达

目的:探讨T细胞淋巴瘤侵袭转移诱导因子l(Tiam1)在动情周期中小鼠子宫内膜的表达规律。方法:采用RT-PCR、免疫组织化学法和Western blotting分别检测小鼠子宫内膜在动情前期、动情期、动情后期和动情间期Tiam1mRNA和蛋白的表达规律。结果:在动情周期中,小鼠子宫内膜mRNA在动情期表达最高,与其它3期相比具有显著性差异,P<0.01;免疫组织化学和Westernblotting的结果也显示Tiam1蛋白在动情期子宫内膜的表达规律与RT-PCR的结果一致。结论:Tiam1在小鼠动情周期子宫内膜中呈动态表达,提示Tiam1可能参与了动情周期子宫内膜变化的调控。     

iNOS和caspase 3在胚胎停育绒毛和蜕膜组织中的表达

目的：探讨胚胎停育绒毛组织和蜕膜组织中半胱氨酸天冬氨酸蛋白酶3（caspase 3）和诱导型一氧化氮合酶（inducible nitric oxide synthase,iNOS）的表达和临床意义。方法：选择2013年3月-2014年5月就诊于武警后勤学院附属医院妇产科计划生育门诊的30例不明原因早期胚胎停育的患者（病例组）和30例正常早期妊娠妇女（对照组）为研究对象,采用逆转录聚合酶链反应（RT-PCR）方法检测2组绒毛组织和蜕膜组织中caspase 3和iNOS mRNA的表达水平,采用免疫印迹法检测2组绒毛组织和蜕膜组织中caspase 3蛋白的表达水平。结果：①病例组绒毛组织和蜕膜组织中caspase 3 mRNA、总的caspase 3蛋白和活化的caspase 3蛋白的表达水平均高于对照组（P＜0.01）。②病例组绒毛组织和蜕膜组织中iNOS mRNA表达水平低于对照组（P＜0.01）。结论：正常早孕和胚胎停育患者绒毛组织和蜕膜组织中均有iNOS和caspase 3的表达,iNOS和caspase 3可能参与凋亡的增加进而引起胚胎停育的发生。     

一氧化氮和一氧化氮合酶在宫内窒息胎鼠肝损伤中的作用及意义

一氧化氮 (nitric oxide,NO )作为一种重要的细胞内信使 ,具有多种生理功能。一氧化氮合酶 (nitric oxide syn-thase,NOS )是合成 NO的唯一限速酶。肝组织中存在内皮型 (endothelial NOS,e NOS)和诱导型 (inducible NOS,i-NOS) ,其诱导产生的 NO参与生理和病理变化。但在宫内窒     

儿茶酚邻位甲基转移酶基因在小鼠动情周期和早孕期间的表达及调控

目的:探讨儿茶酚邻位甲基转移酶(Comt)基因在小鼠动情周期中的表达调控规律,为其参与调节生殖过程的作用机制提供新的证据。方法:采用RT-PCR和实时定量PCR的方法检测成年小鼠动情周期中不同时相以及孕d7的子宫、卵巢组织Comt mRNA水平变化;性成熟前小鼠分别用E2(苯甲酸雌二醇)、P4(孕酮)、E2+Tam(苯甲酸雌二醇+他莫昔芬)、P4+RU486(孕酮+米非司酮)处理,检测其子宫和卵巢组织的mRNA水平。结果:成年小鼠动情期子宫、卵巢组织ComtmRNA水平较高,与动情前期、动情后期、动情间期相比,差异有统计学意义(P<0.05);虽然性成熟前小鼠子宫组织的各处理组与对照组间无显著性差异(P>0.05),但卵巢组织的处理组与对照组间有统计学差异(P<0.05)。卵巢组织中Comt mRNA水平E2处理组显著低于对照组,而E2+Tam组显著高于E2处理组(P<0.05)。妊娠组子宫中Comt mRNA水平显著低于未孕组(P<0.05),而卵巢组织的组间差异不显著(P>0.05)。结论:小鼠Comt mRNA水平随动情周期变化,且与妊娠有关;雌激素对Comt的生物合成具有负调节作用,而且这种作用可能是通过雌激素受体依赖性方式进行的;但是孕激素的作用不显著。     

Localization of granulocyte-macrophage colony-stimulating factor in the bovine reproductive tract

Granulocyte-macrophage colony-stimulating factor (GM-CSF) can increase embryo development to the blastocyst stage in cattle. The objective of the present study was to determine whether GM-CSF is present in the reproductive tract. Using Western blotting, immunoreactive GM-CSF was detected in uterine flushings from cows at days 0, 7, and 14 of the estrous cycle and from cows at days 14-17 of pregnancy. Also, GM-CSF was localized immunohistochemically to endometrium and oviduct. Patterns of immunohistochemical localization and intensity of reaction product were similar for all days of the estrous cycle. While present in several cell types, immunoreactive product in the endometrium was greatest in epithelium (especially luminal epithelium). Immunoreactive GM-CSF was also localized to epithelium in ampullary and isthmic regions of the oviduct, with intensity greater in ampulla. Staining was observed for both ciliated and non-ciliated cells. In conclusion, the bovine oviduct and endometrium contain immunoreactive GM-CSF and this molecule is present in uterine secretions. Thus, this cytokine is a potentially important intracellular regulator of endometrial, oviductal and embryonic function during early pregnancy in the cow.     

Cytokine-induced expression of nitric oxide synthases in Chlamydia trachomatis-infected spontaneous aborters

Purpose: The aim of study was to evaluate expression of inducible nitric oxide synthase (iNOS), endothelial nitric oxide synthase (eNOS), interferon-gamma (IFN-γ), and tumor necrosis factor-alpha (TNF-α) in Chlamydia trachomatis (CT)-infected spontaneous aborters (SA).

Materials and methods: Endometrial curettage tissue was collected from 140 SA (sporadic SA- 70; recurrent SA- 70) (Group I) and 140 age-matched controls (Group II) from Department of Obstetrics and Gynecology, Safdarjung Hospital, New Delhi, India. Polymerase chain reaction was performed for diagnosis of CT. The expression of iNOS/ eNOS/ IFN-γ/ TNF-α was assessed by real-time polymerase chain reaction (PCR).

Results: 15.7% SA were CT-positive (Group I); none in controls. Sporadic spontaneous aborters (SSA) (n?=?8/70), recurrent spontaneous aborters (RSA) (n?=?14/70) diagnosed as CT-positive (Group-I). Significant upregulation of iNOS/ eNOS was found in CT-positive SSA/RSA compared with CT-negative SSA/RSA and healthy controls. TNF-α and IFN-γ were expressed in CT-positive SSA/RSA compared with negative SSA/controls. iNOS showed a significant strong positive correlation with TNF-α and IFN-γ in CT-infected SA. eNOS showed a significant positive correlation with TNF-α and no correlation with IFN-γ in CT-infected SA. TNF-α was positively correlated with IFN-γ.

Conclusions: Significantly high expression of iNOS/ eNOS and proinflammatory cytokines affected pregnancy in CT-infected RSA, thereby implying that there occurs cytokine-induced expression of nitric oxide synthase (NOS).     

内皮型一氧化氮合酶mRNA在人类胎盘中的表达

探讨内皮型一氧化氮合酶ｍＲＮＡ在胎盘,脐带中的表达,及ｅＮＯＳ在胎盘中的定位。方法采用原位聚合酶链反应与原位杂交的方法,以１０例正常足月妊娠的胎盘及挤带中的ｅＮＯＳｍＲＮＡ的表达进行定位研究。结论ｅＮＯＳ主要定位于胎盘合体滋养细胞及干绒毛血管内皮细胞。     

Murine pre-eclampsia induced by unspecific activation of the immune system correlates with alterations in the eNOS and AT1 receptor expression in the kidneys and placenta

It remains arguable if an animal model can be of use in pre-eclampsia (PE) studies, as it is clearly a human disease not observed spontaneously in other species. The aim of this study was to investigate whether PE-like signs in mice inoculated with activated Th1 cells were accompanied by abnormal expression of molecules related to the regulation of blood pressure, viz. nitric oxide synthase enzymes (eNOS and iNOS) and angiotensin (Ang) II receptors (AT1R and AT2R), in order to analyse the relevance of this model for human disease. In this model, C57/BL6-mated BALB/c females received lymphocytes crosslined with anti-CD3 and cultured with interleukin (IL)-2 and IL-12 to mimic PE pathology. Control mice received PBS. eNOS, iNOS and AT1R but not AT2R expression was augmented in the kidneys of PE-mice compared with control pregnant mice. The expression of eNOS but not of iNOS was augmented at the fetal-maternal interface of PE-mice as compared with the controls. NOSs regulate the synthesis of NO, a blood pressure and parturition mediator. As its expression is increased in PE patients, our data suggest that the Th1 cells-induced signs in this model are due to similar mechanisms as in humans. AT1R and AT2R mediate the effect of Ang II, and particularly the AT1R appears to be involved in the pathogenesis of human PE. The increased AT1R expression in the kidneys of PE-mice reinforces the theory that Th1 cells elicit a pathological situation closely resembling the human PE. All together, our data support the use of this animal model to study mechanisms underlying clinically overt PE.     

急性缺血缺氧及再灌注时胎鼠肾脏一氧化氮合酶体系的表达

目的　通过研究急性缺血缺氧及再灌注时胎鼠肾脏一氧化氮 (NO)的变化规律及与一氧化氮合酶 (NOS)的关系 ,初步探讨急性缺血缺氧及再灌注时肾损伤的发病机制。　方法　胎龄 2 1日胎鼠为研究对象 ,分为缺血缺氧 10min、3 0min及缺血缺氧 3 0min再灌注 3 0min、2h、6h和 2 4h组 ,应用硝酸还原酶法、免疫组化和RT PCR技术检测不同时间NO含量改变和NOS(内皮型eNOS和诱导型iNOS)活性及基因表达。　结果　随缺血缺氧时间延长NO水平明显降低 ,缺血 10min (2 2 .65± 4.0 1) ,缺血 3 0min(17.0 4± 2 .99)与假手术组 (2 5 .2 6± 3 .0 5 )相比 ,差异有统计学意义 (P <0 .0 1)。随再灌注时间延长NO水平呈双向改变 ,于再灌注 6h升高达高峰 (2 9.78± 2 .18) ,再灌注 2 4h仍维持较高水平 (2 9.45± 2 .78,P <0 .0 5 )。正常时eNOS和iNOS即位于近曲小管。随缺血缺氧及再灌注时间的延长 ,eNOSmRNA含量逐渐降低 ,eNOS光强度逐渐升高 ,活性逐渐减弱 ,与假手术组相比 ,差异有统计学意义 (P <0 .0 1) ;iNOSmRNA含量及光强度改变与之相反 ,于再灌注 6h后与假手术组差异有统计学意义 (P分别 <0 .0 5和 0 .0 1)。　结论　急性缺血缺氧及再灌注时胎鼠肾脏NO水平呈双相改变 ,参与肾损伤 ,其动态变化过程是NOS体系综合作用的结果     

Ethanol exposure induces oxidative stress and impairs nitric oxide availability in the human placental villi: a possible mechanism of toxicity

OBJECTIVE: We undertook this investigation to explore the effects of ethanol exposure on nitric oxide synthase levels and nitric oxide release. Our hypothesis was that ethanol exposure modifies nitric oxide activity within the placenta as a result of oxidative stress. STUDY DESIGN: Four 10-g samples of term normal human placental villous tissue were perifused with nonrecirculating Dulbecco's modified Eagle's medium and 25-mmol/L N-[2-hydroxyethyl]piperazine-N'-[2-ethanesulfonic acid] with 0-, 50-, 100-, or 200-mmol/L ethanol. After 2 hours of exposure, tissue was removed, fixed, and frozen for analysis. Immunohistochemical analysis was performed for subtype I or neuronal nitric oxide synthase (nNOS), subtype II or inducible nitric oxide synthase (iNOS), and subtype III or endothelial nitric oxide synthase (eNOS) localization. Western blot analysis was performed for eNOS quantitation. Cyclic guanosine monophosphate and copper-zinc superoxide dismutase levels were measured by electroimmunoassay and kinetic assay, respectively. Nitric oxide release was analyzed by a Sievers nitric oxide analyzer. RESULTS: Immunohistochemical examination confirmed that only eNOS was localized to the syncytiotrophoblasts. After ethanol exposure, eNOS protein expression increased 2.5- to 3.0-fold over that of the control. Tissue cyclic guanosine monophosphate content and nitric oxide release into the effluent were decreased, whereas superoxide dismutase levels were increased at higher ethanol levels (P <.05). CONCLUSION: Ethanol exposure appears to induce oxidative stress, which may account for the decreased nitric oxide release, because nitric oxide may be shunted toward scavenging free radicals. Increased eNOS protein expression may be a response to the increased demand for nitric oxide. Decreased nitric oxide availability could adversely affect placental blood flow regulation, which could, in turn, account for the growth restriction seen in ethanol-exposed fetuses.     

Expression of endothelial nitric oxide synthase by extravillous trophoblast cells in the human placenta

Previous reports have documented the expression of endothelial nitric oxide synthase (eNOS) expression by the syncytiotrophoblast layer of the villus in the human placenta. In contrast, the underlying villous cytotrophoblast cells do not express the enzyme. Because extravillous cytotrophoblasts have not been as extensively investigated, our objective was to test whether these cells express eNOS. Using both a mouse monoclonal and a rabbit polyclonal antibody, we demonstrated immunoreactive eNOS in trophoblast cell columns emanating from anchoring villi in second trimester placentae. Cytokeratin positive trophoblast cells lying beneath remnant anchoring villi, lining decidual blood vessels and scattered throughout the basal plate of normal term and pre-eclamptic placentae also expressed immunoreactive eNOS. By Western analysis, the monoclonal and polyclonal antibodies were shown to be absolutely and relatively specific for eNOS, respectively. The finding of immunoreactive eNOS expression by extravillous trophoblast cells was substantiated by in situ hybridization. Using riboprobes generated from a bovine eNOS cDNA, we demonstrated specific hybridization in the endothelium of blood vessels in the umbilical cord, thus validating the in situ hybridization methodology, as well as specific hybridization in the extravillous trophoblast cells of the basal plate in normal term placenta. In conclusion, several different populations of extravillous trophoblast cells in the basal plate of the human placenta express eNOS.     

Identification of arginase in human placental villi

l-Arginine is the common substrate for arginase and nitric oxide synthase (NOS). Arginase converts l-arginine to urea and ornithine, which is the principal precursor for production of polyamines required for cell proliferation. Human placenta expresses endothelial NOS (eNOS) in syncytiotrophoblasts, but the expression of arginase has not been fully elucidated. Our aim was to investigate the expression and distribution patterns of arginase-I (A-I) and arginase-II (A-II) in human placental villi in the first trimester and at term using immunohistochemistry, RT-PCR and Western blot analysis. The arginase enzyme activity in placental villi was also measured. Immunohistochemistry showed different distribution patterns of the arginase isoforms during gestation: A-I was observed only in cytotrophoblasts, while A-II was observed in both cytotrophoblasts and syncytiotrophoblasts. RT-PCR and Western blot analysis showed expression of A-I and A-II in the first trimester and at term in human placental villi. Expression of A-II and arginase activity was greater in the first trimester than at term. Differentiation of cytotrophoblasts into syncytiotrophoblasts may be associated with l-arginine metabolism through modulation of l-arginine availability for eNOS and A-I. And elevated arginase activity in the early gestational period may be responsible for proliferation of trophoblasts by increasing polyamines production. These results suggest that the l-arginine-ornithine-polyamine and l-arginine-nitric oxide pathways play a role in placental growth and development.