诱导型一氧化氮合酶与移植血管平滑肌细胞增生的关系

目的　探讨诱导型一氧化氮合酶 (iNOS)与移植血管平滑肌细胞增生的关系。方法　新西兰大白兔 15只随机分成 3组 ,建立双侧颈动脉间置移植颈外静脉的动物模型。高剂量组每日喂食L 精氨酸 (L Arg) 2 5 0mg/kg ,低剂量组每日喂食L Arg 12 5mg/kg ;对照组不喂食L Arg ,持续 2周。检测血浆和组织匀浆一氧化氮(NO)水平 ,移植血管iNOSmRNA表达。观察术后 3和 6周移植血管平滑肌细胞增生。结果　 1.iNOS活性 :(1)血浆NO水平 :实验组血浆NO水平显著高于对照组 ,高剂量组血浆NO水平高于低剂量组 ;(2 )组织匀浆一氧化氮合酶 (NOS)活性 :实验组组织匀浆NOS活性显著高于对照组 ;(3)组织匀浆iNOSmRNA表达 :术后 3周实验组iNOSmRNA表达 ,对照组无表达 ;术后 6周实验组iNOSmRNA表达高于对照组 ,高剂量组高于低剂量组。 2 .移植血管平滑肌细胞形态学变化 :(1)SMA免疫组化染色 :实验组移植血管平滑肌细胞厚度低于对照组 ;术后 6周低剂量组移植血管平滑肌细胞厚度低于高剂量组 ;(2 )PCNA免疫组化染色 :术后 3周实验组平滑肌细胞增殖低于对照组 ;术后 6周低剂量组平滑肌细胞增殖低于对照组和高剂量组。结论　iNOS表达致体内NO水平增高可抑制移植血管平滑肌细胞增生 ;NO浓度与平滑肌细胞增生关系密切     

诱生型一氧化氮合酶特异性抑制肽的制备

目的：通过噬菌体展示技术筛选iNOS特异性抑制肽。方法：将iNOSFAD结合区及其附近序列的基因片段装入pET-28A( )，在大肠杆菌BL21中表达，His.bind^TM亲和层析柱纯化目的蛋白，使用纯化蛋白筛选Ph.D.-12^TM噬菌体库，筛选iNOS活性抑制作用较高的噬菌体克隆，测序并合成其中具有一致序列的短肽。结果：得到具有较高表达量的目的蛋白，经His.Bind^TM柱亲和层析纯化后纯度大于95％，以纯化蛋白筛选Ph.D.-12^TM噬菌体库，经4轮筛选获得10株iNOS活性抑制作用较高的噬菌体克隆，测序发现其中5株序列完全相同，合成该12肽，初步研究表明其对iNOS表现为高浓度抑制，低浓度兴奋的作用，而对nNOS及eNOS则没有影响。结论：以iNOSFAD片段蛋白为靶蛋白筛选得到的克隆对iNOS活性具有特异性影响，可根据这些特征设计合成小分子前导药物，创造新的活性药物。     

Immunohistochemical analysis of inducible nitric oxide synthase (iNOS) and heat shock proteins (HSPs) in ameloblastomas

BACKGROUND: To clarify the possible role of nitric oxide (NO) and stress proteins in oncogenesis and cytodifferentiation of odontogenic epithelium. Inducible NO synthase (iNOS) and heat shock proteins (HSPs) were analyzed in ameloblastomas as well as in tooth germs. METHODS: Specimens of seven tooth germs, 36 benign ameloblastomas and five malignant ameloblastomas were examined by immunohistochemistry using antibodies against iNOS and 27-, 60- and 70-kDa HSPs (HSP27, HSP60 and HSP70). RESULTS: Immunoreactivity for iNOS was detected in normal and neoplastic odontogenic epithelial cells and was higher in malignant ameloblastomas than in tooth germs and benign ameloblastomas. HSP27 was expressed constitutively in all odontogenic epithelial cells in tooth germs and benign and malignant ameloblastomas. Expression of HSP60 and HSP70 was detected in normal and neoplastic odontogenic epithelial cells and was prominent in cells neighboring the basement membrane. HSP60 reactivity showed no apparent difference between normal and neoplastic odontogenic epithelium, whereas HSP70 expression was slightly higher in benign and malignant ameloblastomas than in tooth germs. CONCLUSIONS: Activation of iNOS might be associated with malignant potential of epithelial odontogenic tumors. Elevated expression of HSP70 is considered to be involved in neoplastic transformation of odontogenic epithelial cells.     

肠缺血/再灌注致肺损伤时肺内HO-1/CO与iNOS/NO相互作用的研究

目的观察肠缺血/再灌注(I/R)致肺损伤时肺内HO-1/CO与iNOS/NO的相互作用。方法采用肠缺血/再灌注模型。32只Wistar大鼠随机分为假手术组(Sham组)、肠缺血1 h再灌注6 h组(I/R组)、氨基胍组(AG组)和血晶素组(hemin组)。检测肺组织中HO-1和iNOS的表达,观察肺组织丙二醛(MDA)、血清一氧化氮(NO)及动脉血中氧血红蛋白(Hb-CO)的含量,同时观察肺组织病理形态学改变。结果与Sham组比较,I/R组HO-1和iNOS表达显著增强(均P<0.01);AG组HO-1和iNOS表达较I/R组明显降低(均P<0.05);Hemin组iNOS表达较I/R组明显降低而HO-1表达明显升高(均P<0.05);I/R组肺组织MDA、血清NO、血中HbCO较Sham组显著增加(P<0.05或P<0.01);与I/R组比较,AG组、Hemin组肺组织MDA、血清NO显著降低(P<0.05或P<0.01)。AG组的HbCO明显降低而Hemin组的HbCO明显升高(P<0.05)。病理学检查显示,AG组与Hemin组肺组织损伤程度较I/R组明显减轻。结论NO及CO对肠I/R肺组织具有保护作用,两者之间存在着相互作用,肺内HO-1/CO的大量生成具有使NO产生减少的作用,同时iNOS/NO的过量生成具有上调HO-1表达使CO产生增多的作用。     

An animal model of chronic inflammatory pain: pharmacological and temporal differentiation from acute models.

Clinically, inflammatory pain is far more persistent than that typically modelled pre-clinically, with the majority of animal models focussing on short-term effects of the inflammatory pain response. The large attrition rate of compounds in the clinic which show pre-clinical efficacy suggests the need for novel models of, or approaches to, chronic inflammatory pain if novel mechanisms are to make it to the market. A model in which a more chronic inflammatory hypersensitivity phenotype is profiled may allow for a more clinically predictive tool. The aims of these studies were to characterise and validate a chronic model of inflammatory pain. We have shown that injection of a large volume of adjuvant to the intra-articular space of the rat knee results in a prolonged inflammatory pain response, compared to the response in an acute adjuvant model. Additionally, this model also results in a hypersensitive state in the presence and absence of inflammation. A range of clinically effective analgesics demonstrate activity in this chronic model, including morphine (3mg/kg, t.i.d.), dexamethasone (1mg/kg, b.i.d.), ibuprofen (30mg/kg, t.i.d.), etoricoxib (5mg/kg, b.i.d.) and rofecoxib (0.3-10mg/kg, b.i.d.). A further aim was to exemplify the utility of this chronic model over the more acute intra-plantar adjuvant model using two novel therapeutic approaches; NR2B selective NMDA receptor antagonism and iNOS inhibition. Our data shows that different effects were observed with these therapies when comparing the acute model with the model of chronic inflammatory joint pain. These data suggest that the chronic model may be more relevant to identifying mechanisms for the treatment of chronic inflammatory pain states in the clinic.     

一氧化氮与胚胎异常发育的相关性研究

为了解开一氧化氮（ＮＯ）是否与畸胎发生有关这一谜团和进一步阐明砷致畸作用机理，本实验应用诱生型ＮＯ合成酶（ｉＮＯＳ）组织化学、扫描电镜（ＳＥＭ）及体内致畸试验等方法研究了砷对小鼠卵黄囊胎盘（ＹＳＰ）和胚胎发育的影响。结果表明ＹＳＰ细胞ｉＮＯＳ表达与砷浓度之间存在明显的剂量—反应关系（Ｐ＜０．０５）；ＳＥＭ观察可见ＹＳＰ内皮层和间皮层细胞受损；光镜下可见ＹＳＰ变小、萎缩和微血管分化不良；随着染毒剂量的升高，畸胎率和死胎率亦逐步增加，最高分别达到５６．８％和２４．７％；畸胎的主要表现是神经管未闭，心包积液和体位异常等。研究结果率先提示过量ＮＯ与畸胎发生及致畸机理关系密切；推荐在致畸研究中ｉＮＯＳ可作为一种有效的生物标志物。     

Short-circuiting autoimmune disease by target-tissue-derived nitric oxide

A previous report from this laboratory suggested that expression of skeletal-muscle-derived, inducible nitric oxide synthase (iNOS), is associated with resistance to the autoimmune model of myasthenia gravis (MG) demonstrated by Wistar Furth rats following the passive transfer of antibody reactive with the nicotinic acetylcholine receptor (AChR). The study reported below demonstrates an association between increased expression of iNOS/NO in Wistar Furth rats and the induction of programmed cell death (apoptosis) in both macrophages and CD4+ T cells that attempt to traffic through targeted muscles. It is concluded that production of muscle-derived NO is protective in experimental MG, and in part, dictates the severity of eventual immunopathology.     

精索内动静脉高位结扎iNOS的表达与睾丸生精细胞增殖和凋亡的关系

目的：探讨精索内动静脉高位结扎术后，诱导型一氧化氮合酶（inducible nitric oxide synthase．iNOS）的表达与生精细胞增殖和凋亡的关系。方法：免疫组化结合计算机图像分析系统检测iNOS阳性细胞光密度值；免疫组化方法检测并计算PCNA增殖指数（PCNA-Labeling index，PI）；原位末端标记法（terminal deoxynucleotidyl transferase mediated dUTP nick-endlabeling method，TUNEL）检测生精细胞凋亡指数（apoptosis index，AI）。分析iNOS的表达与增殖、凋亡的相关性。结果：iNOS在术后3-15d表达增强，第7d呈强阳性表达。结扎组生精细胞PCNA增殖指数（PI）明显低于对照组（P〈0．01），凋亡细胞指数（AI）明显高于对照组（P〈0．01）。结论：精索内动静脉高位结扎，iNOS的表达与生精细胞PCNA增殖指数呈负相关，与生精细胞凋亡指数呈正相关。iNOS基因表达增强是生精细胞增殖减少和凋亡增加的重要原因。     

新生鼠脑损伤后松果体细胞的一氧化氮合酶与细胞凋亡关系的初步研究

为了探讨新生鼠发生缺氧缺血性脑损伤时松果体细胞的诱生型一氧化氮合酶 ( i NOS)表达与松果体细胞凋亡及形态学改变的关系 ,用 7日龄新生 Wistar大鼠 ,结扎左侧颈总动脉 ,术后 2 h吸入 8%浓度氧 2 h,建立新生儿缺氧缺血性脑病模型( HIBD)。分别于建模后 0 h、2 4h、48h处死动物 ,剥取松果体 ,观察松果体细胞的 i NOS表达及细胞凋亡 ,电镜观察松果体形态学改变。结果表明 :( 1)新生鼠脑损伤后松果体的 i NOS在 0 h、2 4h含量与对照组相比明显升高 ;( 2 )脑损伤后松果体凋亡细胞早期明显增多 ,尤以 0 h、2 4h为主 ;( 3)电镜观察 :脑损伤后松果体的形态学也以 0 h、2 4h改变为明显 ,出现线粒体明显肿胀、粗面内质网极度扩张、细胞变性。提示 :( 1)新生鼠缺氧缺血性脑损伤后 0 h、2 4h松果体细胞的 i NOS表达增加 ,以后逐渐下降 ,48h表达减少。 ( 2 ) TUNEL法原位检测细胞凋亡与 i NOS表达同步改变 ,i NOS表达对细胞凋亡有促进作用。 ( 3) i NOS表达和细胞凋亡参与了新生鼠缺氧缺血性脑损伤后松果体细胞形态学改变     

IFN-γ在沙眼衣原体呼吸道感染中免疫防御机制的探讨

目的:检测与IFN-γ作用相关的酶吲哚胺2,3二氧化酶(IDO)、诱导性一氧化氮合成酶(iNOS)和NADPH氧化酶(ox)gp91在沙眼衣原体呼吸道感染中的表达及与机体防御的关系,探讨衣原体感染中IFN-γ免疫防御作用的机制.方法:用沙眼衣原体小鼠肺炎株(MoPn)通过鼻腔感染C57BL/6(H-2b)小鼠,用过氧化物酶连接的鼠抗衣原体脂多糖单抗染色HeLa 229细胞,检测衣原体在肺组织的生长;用RT-PCR检测衣原体感染后第7及14天小鼠肺组织IFN-γ、IDO、iNOS和gp91NADPH ox mRNA表达.结果:MoPn呼吸道感染后小鼠肺组织匀浆衣原体活性测定,于感染后第2天,HeLa 229细胞内可见有衣原体包涵体生长,IFU值增高,于感染后第7天IFU达最高水平,以后逐渐下降,至感染后21天基本恢复到基线水平.与未感染的对照组比较,Th1细胞因子IFN-γ于感染后第7天表达显著增高,感染后14天有所降低,但仍维持较高水平;同时衣原体感染可显著诱导与IFN-γ作用相关的三种酶IDO、iNOS和gp91 NADPH ox在小鼠肺组织的表达,感染后第7及14天,IDO,iNOS及gp91 NADPH ox的表达与对照组比较均有显著差异,其中IDO和gp91 NADPH ox于感染后第7天mRNA表达增高显著(P＜0.01),14天略有下降(P＜0.05).结论:衣原体呼吸道感染诱导Th1细胞因子IFN-γ mRNA高表达,参与宿主对衣原体的清除及机体免疫防御,此作用可能与其相应的酶IDO、iNOS和gp91 NADPH ox表达增高有关.