bcl—2基因反义核酸对白血病细胞药物敏感性的影响

目的：探讨ｂｃｌ－２基因反义核酸对白血病细胞药物敏感性的影响。方法：应用细胞培养，免疫标记及流式细胞仪技术，检测了ｂｃ１－２基因反义寡脱氧核糖核苷酸（ＡＳＯＤＮ）和阿糖胞苷（Ａｒａ－Ｃ）对白血病Ｋ５６２和ＨＬ－６０细胞作用和ｂｃｌ－２基因表达。结果：Ａｒａ－Ｃ（１０μｍｏｌ／Ｌ）与ｂｃｌ－２ＡＳＯＤＮ同时作用比单独用Ａｒａ－Ｃ或Ａｒａ－Ｃ与正义寡脱氧核糖核苷酸（ＳＯＤＮ）细胞存活率显著减少（Ｐ＜０００１），并且流式细胞仪检测显示ｂｃｌ－２ＡＳＯＤＮ使ｂｃｌ－２蛋白阳性率降低。结论：ｂｃｌ－２基因反义寡核苷酸能抑制该基因表达，提高白血病细胞对Ａｒａ－Ｃ的敏感性     

诱导型一氧化氮合酶在强啡肽致脊髓损伤中的作用

目的：探讨诱导型一氧化氮合酶（ｉＮＯＳ）在强啡肽致脊髓损伤中的作用。方法：［３Ｈ］－左旋精氨酸转化法测定腹侧和背侧脊髓ｉＮＯＳ活性,原位杂交法观测脊髓ｉＮＯＳｍＲＮＡ表达及其细胞分布。结果：大鼠蛛网膜下腔注射（ＩｎＩ）强啡肽Ａ１－１７（Ｄｙｎ）２０ｎｍｏｌ引起持久性截瘫和迟发性神经元死亡;在Ｄｙｎ致瘫后２～３ｈｉＮＯＳｍＲＮＡ表达开始增多增强,４ｈ达高峰,２４ｈ和４８ｈ仍见广泛表达,其分布以胶质细胞和大运动神经元为主;腹侧脊髓ｉＮＯＳ活性在Ｄｙｎ致瘫后４ｈ显著升高,并持续至２４ｈ和４８ｈ;提前１０ｍｉｎＩｎＩ选择性ｉＮＯＳ抑制剂氨基胍１μｍｏｌ可显著对抗Ｄｙｎ２０ｎｍｏｌ引起的持久瘫及伤后４ｈ腹侧脊髓ｉＮＯＳ活性升高。结论：ｉＮＯＳ持续性高表达与Ｄｙｎ致脊髓损伤机制有关     

小儿哮喘发病中CD23,IFN和IgE的作用及其意义

应用ＡＰＡＡＰ方法，Ｄｏｔ－ｂｌｏｔ地高标记核酸杂交技术及ＡＢＣ－ＥＬＩＳＡ法分别检测哮喘患巴细胞ＣＤ２３ｍＲＮＡ，膜ＣＤ２３分子表达和患儿血浆和淋巴细胞诱生ＩＦＮ－γ水平。结果，哮喘患儿ＣＤ２３ｍＲＮ表达增高，外周血ＰＢＭＣ和Ｂ细胞的ＣＤ２３表达均显著增加，ＩＦＮ－γ诱生水平明显低下，血清ＩｇＥ水平异常升高，Ｂ细胞ＣＤ２３分子表达与ＩｇＥ水平之间显著正相关麻疹疫苗治疗后Ｂ细胞ＣＤ２３分子表达明显     

针刺对小鼠腹腔巨噬细胞的热休克蛋白、诱导性一氧化氮合酶及其基因表达的效应

目的研究针刺对小鼠腹腔巨噬细胞的热休克蛋白（ＨＳＰ）、诱导性一氧化氮合酶（ｉＮＯＳ）及其ｍＲ－ＮＡ表达的效应。方法将２４只昆明小鼠腹腔注射无菌石蜡油后,随机分为３组：电针（ＥＡ）组、对照１（Ｃ１）组和对照２（Ｃ２）组。将３组收集的腹腔巨噬细胞制备成玻片和硝酸纤维素膜（ＮＣＭ）两种标本,应用原位杂交、免疫细胞化学、细胞化学及斑点印迹技术检测ＨＳＰ７０、ｉＮＯＳ及ｉＮＯＳｍＲＮＡ。结果ＨＳＰ７０定位于巨噬细胞的胞质和胞核;ｉＮＯＳｍＲＮＡ及ｉＮＯＳ均定位于巨噬细胞的胞质;３组的ｉＮＯＳｍＲＮＡ、ｉＮＯＳ、ＨＳＰ７０的斑点印迹的扫描数值均为ＥＡ组＞Ｃ２组＞Ｃ１组（Ｐ＜０．０１）。结论电针可显著提高小鼠腹腔巨噬细胞的ＨＳＰ７０、ｉＮＯＳ及ｉＮＯＳｍＲ－ＮＡ表达。     

一氧化氮在培养的大鼠心肌细胞缺氧—复氧损伤中的作用

目的：观察一氧化氮（ＮＯ）对心肌细胞缺氧－复氧损伤（ＨＲＩ）的作用。方法：培养的大鼠心肌细胞，培养液中分别预先加入ＮＯ前体Ｌ－精氨酸（Ｌ－Ａｒｇ）、ＮＯ供体ＳＩＮ－１或硝普钠（ＳＮＰ）、ＮＯＳ抑制剂Ｌ－ＮＮＡ或ＮＯＳ诱导剂脂多糖（ＬＰＳ），经缺氧１２０ｍｉｎ，复氧６０ｍｉｎ处理后，检测细胞存活率，乳酸脱氢酶（ＬＤＨ）漏出量，亚硝酸盐（ＮＯ２）含量及细胞诱导型ＮＯ合酶（ｉＮＯＳ）活性等指标的改变。结果：①与常氧组比较，缺氧－复氧ＨＲ降低细胞存活率（２３％，Ｐ＜０．０１），增加ＬＤＨ漏出（６２倍，Ｐ＜０．０１），ｉＮＯＳ活性（７７％，Ｐ＜０．０１），ＮＯ２含量（６１７％，Ｐ＜０．０１）。②ＨＲ前预先加入ＳＩＮ－１、ＳＮＰ或Ｌ－Ａｒｇ，均引起ＬＤＨ漏出进一步增高（Ｐ＜０．０１），细胞存活率进一步降低（Ｐ＜０．０１或Ｐ＜０．０５）。③Ｌ－ＮＮＡ２ｍｍｏｌ／Ｌ，单独应用对细胞损伤的影响无统计学意义，与Ｌ－Ａｒｇ联合应用，则减弱Ｌ－Ａｒｇ的细胞损伤作用。④ＬＰＳ１μｇ／ｍＬ，增加ｉＮＯＳ活性（２６倍，Ｐ＜０．０１）和ＬＤＨ漏出（５６％，Ｐ＜０．０１）。结论：ＮＯ加重心肌细胞ＨＲＩ，是心肌细胞ＨＲＩ的损伤因子     

β—内啡肽增强人外周血单个核细胞IL—1β,IL—6和 i …

目的 研究β－内啡肽（β－ｅｎｄｏｒｐｈｉｎ,β－ＥＮＤ）对人外周血单个核细胞（ＰＢＭＣ）细胞因子ＩＬ－１β、ＩＬ－６和诱导型一氧化氮合酶（ｉＮＯＳ）的ｍＲＮＡ表达,探讨神经递质对免疫细胞产生细胞因子和ｉＮＯＳ的调节作用。方法 以不同浓度β－ＥＮＤ体外作用于人外周血单个核细胞,随后提取细胞ＲＮＡ。逆转录成ｃＤＮＡ,并分别和相应的上下游引物进行ＰＣＲ扩增,扩增产物在含有溴化乙锭（ＥＢ）的琼脂糖凝胶上     

细胞性一氧化氮供体的建立及其对肿瘤细胞凋亡的诱 …

一氧化氮供体细胞系的建立及其对肿瘤细胞凋亡的诱导效应研究。方法采用＾３Ｈ－胍氨酸转移法进行了ＮＯ合成酶活性测定，亚硝酸盐检测；ＤＮＡ片段的提取及凝胶电泳分析；凋亡细胞的ＤＡＰ１染色观察；Ｗｅｓｔｅｒｎ ｂｌｏｔ分析。结果将ｉＮＯＳ真核表达质粒，ｐＣＭＶ／ｉＭＯＳ转染至Ｓｐ２／０骨髓瘤的变异株中，并获得能稳定表达ｉＮＯＳ和合成ＮＯＲ 重组细胞系，表达ｉＮＯＳ的效率为每升培养物含４００μｇ蛋白。     

β-内啡肽增强人外周血单个核细胞IL-1β、IL-6和iNOS mRNA表达

目的　研究β内啡肽（βｅｎｄｏｒｐｈｉｎ ，βＥＮＤ） 对人外周血单个核细胞（ＰＢＭＣ） 细胞因子ＩＬ１β、ＩＬ６ 和诱导型一氧化氮合酶（ｉＮＯＳ） 的ｍ ＲＮＡ 表达， 探讨神经递质对免疫细胞产生细胞因子和ｉＮＯＳ 的调节作用。方法　以不同浓度βＥＮＤ 体外作用于人外周血单个核细胞，随后提取细胞ＲＮＡ。逆转录成ｃＤＮＡ，并分别和相应的上下游引物进行ＰＣＲ 扩增，扩增产物在含有溴化乙锭（ＥＢ）的琼脂糖凝胶上电泳，泳动带经激光密度扫描仪扫描进行定量。结果　βＥＮＤ 明显增强人外周血单个核细胞ＩＬ１β、ＩＬ６ 和ｉＮＯＳ的ｍ ＲＮＡ 表达。结论　神经肽对免疫功能的调节包括调节免疫细胞产生细胞因子和ｉＮＯＳ。     

细胞性一氧化氮供体的建立及其对肿瘤细胞凋亡的诱导效应

目的一氧化氮（ＮＯ）供体细胞系的建立及其对肿瘤细胞凋亡的诱导效应研究。方法采用３Ｈ－胍氨酸转换法进行ＮＯ合成酶（ＮＯＳ）活性测定；亚硝酸盐检测；ＤＮＡ片段的提取及凝胶电泳分析；凋亡细胞的ＤＡＰＩ染色观察；Ｗｅｓｔｅｒｎｂｌｏｔ分析。结果将ｉＮＯＳ真核表达质粒、ｐＣＭＶ／ｉＮＯＳ转染至Ｓｐ２／０骨髓瘤的变异株中，并获得能稳定表达ｉＮＯＳ和合成ＮＯ的重组细胞系（ＳＰｍｔ／ｉＮＯＳ），表达ｉＮＯＳ的效率为每升培养物含４００μｇ蛋白。利用该细胞作ＮＯ细胞性供体，成功地证实ＮＯ能诱导Ｓｐ２／０骨髓瘤细胞发生凋亡。结论所建细胞性ＮＯ供体应用于ＮＯ的生物学功能研究具有ＮＯ合成稳定；更能模拟体内细胞间ＮＯ的信息传递过程；不需任何外源刺激即可合成ＮＯ等独特优点。所建ＮＯ供体细胞可以用于肿瘤细胞凋亡的研究。     

宫颈癌浸润淋巴细胞和Langerhans细胞免疫组织化学研究

目的：研究宫颈癌浸润淋巴细胞、Ｌａｎｇｅｒｈａｎｓ细胞分布及与淋巴细胞活性标记物ＨＬＡ－ＤＲ和转铁蛋白受体（ＴｆＲ）的关系。方法：应用ＣＤ４，ＣＤ８，ＯＫＴ１１，ＯＫＴ９，ＨＬＡ－ＤＲ和Ｓ－１００抗体及ＡＢＣ免疫组化法。结果：ＣＤ４，ＣＤ８，ＯＫＴ１１阳性细胞随肿瘤发展而增多，并和转铁蛋白受体及ＨＬＡ－ＤＲ阳性表达密切关联。ＬＣ细胞数目随肿瘤分期增高和ＴｉＬ密度增另而增多。结论：ＴｉＬ和ＬＣ在宫颈     

葛根素对糖尿病大鼠白内障过程中晶状体诱导型一氧化氮合酶的影响

目的: 观察大鼠糖尿病性白内障(DC)晶状体诱导型一氧化氮合酶(iNOS)基因表达变化及葛根素对DC的治疗作用。方法: 经腹腔注射链脲佐菌素(STZ)复制大鼠DC模型。实验分为STZ组(STZ,45 mg/kg bw,ip)、葛根素组(STZ+葛根素,140 mg/kg bw ip)和对照组(等量生理盐水,ip),分别于实验第20、40、60 d摘取大鼠晶状体,用逆转录聚合酶链反应(RT-PCR)、蛋白印迹(Western blot)和生化方法检测晶状体iNOS mRNA和蛋白表达及一氧化氮(NO)含量、一氧化氮合酶(NOS)活性变化,观察晶状体损伤的宏观和微观病理变化。 结果:对照组大鼠晶状体透明,iNOS mRNA未见明显表达,蛋白呈微弱表达,NOS活性较低,NO含量较少。在DC形成过程中,晶状体出现混浊、晶状体上皮细胞(LEC)有明显病变,并随病程延长而加重;晶状体iNOS mRNA和蛋白表达明显上调,NOS活性增强、NO生成增加,并呈现时间依赖性。用药40-60 d时葛根素组前述晶状体病理变化轻于STZ组,iNOS mRNA和蛋白表达明显低于STZ组,NOS活性及NO 生成低于STZ组。结论: 在大鼠DC形成过程中晶状体iNOS 基因表达上调、NO含量增加;葛根素可减轻晶状体损伤,机制可能与抑制iNOS 基因表达、减少NO生成有关。     

Expression and Localization of Inducible Nitric Oxide Synthase in Anti-Thy-1 Glomerulonephritis

To elucidate a possible involvement of nitric oxide in the development of a mesangial proliferative glomerulonephritis induced by anti-Thy-1 antibody administration, glomerular expression of three isoforms of NO synthase (NOS), inducible NOS (iNOS), brain NOS, and endothelial NOS, was examined at both mRNA and protein levels by ribonuclease protection assay and immunofluorescence microscopy. Light microscopy showed an accumulation of polymorphonuclear leukocytes at 1 hour, lysis of mesangial cells at 1 day, a mesangial proliferative lesion at 4 to 10 days, and minimal residual glomerular lesions by 28 days. Ribonuclease protection assay showed that the glomerular expression of iNOS mRNA peaked at 1 hour and decreased thereafter. No substantial expression of iNOS mRNA was observed in normal glomeruli or in the nephritic glomeruli obtained at different time points (1, 4, 10, or 28 days). By immunofluorescence microscopy with a specific monoclonal antibody, an intense reaction for iNOS was demonstrated in a few cells in the glomeruli at 1 hour. Most of the iNOS-positive cells were identified as polymorphonuclear leukocytes. iNOS-positive cells were found less frequently in the glomeruli on days 1 and 4. Endothelial NOS mRNA was constitutively expressed in normal glomeruli and increased biphasically with two peaks at 1 hour and at 4 days or later; however, the peak expression was much less than that of iNOS mRNA at 1hour. Expression of brain NOS mRNA was not detectable in either normal or nephritic glomeruli. These results show that iNOS is predominantly expressed in polymorphonuclear leukocytes accumulating at 1 hour in the glomeruli of anti-Thy-1 glomerulonephritis and suggest an involvement of NO in the initiation of the disease.     

Inducible nitric oxide synthase expression in coronary arteries of transplanted human hearts with accelerated graft arteriosclerosis.

Inducible nitric oxide synthase (iNOS) is a high-output isoform of NOS that produces nitric oxide (NO), a nonspecific immune effector molecule. In some animal models of autoimmunity, the induction of iNOS has been shown to lead to inflammation and tissue damage, and it has been suggested that iNOS is an immune mediator in humans as well. Using in situ hybridization and immunohistochemical techniques, we demonstrate that iNOS mRNA and protein are present in the coronary arteries of transplanted human hearts with accelerated graft arteriosclerosis (AGA). iNOS is expressed in cells morphologically consistent with macrophages in the neointima of 7 of 10 of the transplanted vessels with AGA that were examined. In serial sections, these same cells express the macrophage marker CD68. In contrast, iNOS is absent from five native coronary arteries with atherosclerosis and absent from two normal coronary arteries. Although iNOS is expressed in macrophages in AGA, its role in the pathogenesis of AGA is unknown.     

Nitric oxide synthase and nitric oxide production in in vivo-derived mast cells

Nitric oxide (NO) is a potent mediator synthesized by a variety of cells involved in inflammatory reactions. We investigated the expression of NO synthase (NOS) in rat peritoneal mast cells (PMC). Small amounts of eNOS mRNA were detected basally, whereas neither mRNA for iNOS nor nNOS was detected in unstimulated PMC. Following stimulation by antigen, interferon-gamma (IFN-gamma), or anti-CD8 antibody, PMC up-regulated iNOS mRNA expression. In situ RT-PCR confirmed that iNOS mRNA originated from PMC. Production of iNOS protein was confirmed in stimulated PMC by immunohistochemistry. Upon stimulation with antigen, IFN-gamma, or anti-CD8, nitrite production was increased significantly (8.4+/-0.6, 7.6+/-0.9, and 6.6+/-0.9 microM/2x10(5) cells/48 h NO2-, respectively; P<0.01), whereas unstimulated PMC released 2.1 +/- 0.3 microM/2 x 10(5) cells/48 h NO2-. These findings demonstrate that in vivo-derived PMC transcribe and translate mRNA for NOS and produce NO.     

体外反搏对心肌梗死犬一氧化氮系统的影响

目的:探讨体外反搏对心肌梗死犬一氧化氮(NO)、一氧化氮合酶(NOS)和其基因表达的影响。方法:19只健康杂种犬随机分为对照组、缺血组和缺血+反搏组(反搏组)3组,采用开胸结扎冠状动脉左前降支的方法建立心肌缺血模型,用改良硝酸还原酶法测定心肌缺血前后血清NO含量、以及心肌组织的NO含量和NOS比活性,采用免疫组化方法检测缺血区心肌组织的NOS亚型即诱导型NOS(iNOS)和内皮型NOS(eNOS)的蛋白合成,用原位杂交方法检测构成型NOS(cNOS)信使核糖核酸(mRNA)的基因表达。结果:在冠状动脉结扎前和结扎后60min,3组犬血清NO含量均无明显差异(P＞0.05);结扎后120min和180min时,反搏组犬血清NO含量明显高于缺血组(P＜0.05)。正常组和反搏组犬心肌组织NO含量和NOS比活性均大于缺血组(P＜0.05)。免疫组化结果表明心肌缺血时iNOS蛋白合成增多,而eNOS蛋白合成减少;体外反搏对iNOS有抑制作用,对eNOS有促进作用。此外心肌缺血时cNOSmRNA的表达明显减少,反搏可促进cNOSmRNA的表达。结论:体外反搏促进NO的产生可能是其抗心肌缺血性损伤的重要机制之一。     

吸烟大鼠一氧化氮合酶和一氧化氮的变化

目的:观察吸烟对大鼠肺组织iNOS、eNOSmRNA和蛋白表达以及支气管肺泡灌洗液(BALF)中NO的影响, 探讨不同类型的NOS在吸烟所致慢性气道炎症中的作用。方法:选用Wistar大鼠80只随机分为对照组, 被动吸烟组, iNOS抑制剂L-NIL干预组及NOS抑制剂L-NAME干预组。用免疫组化法检测iNOS及eNOS的蛋白表达, 用RT-PCR检测iNOS及eNOSmRNA的表达, 用Griess法测定BALF中的NO^-₂/NO^-₃含量。结果:吸烟大鼠肺组织中iNOSmRNA及其蛋白表达增加, eNOSmRNA及蛋白表达下降, BALF中细胞总数及NO^-₂/NO^-₃显著增加(P＜0.05)。在体实验发现, L-NIL使BALF中细胞总数及NO^-₂/NO^-₃下降(P＜0.05);L-NAME对BALF中细胞总数及NO^-₂/NO^-₃无显著影响(P＞0.05)。结论:吸烟大鼠肺组织iNOSmRNA和蛋白表达增加, eNOSmRNA和蛋白表达减少。活化的iNOS产生大量NO促进炎症发展。     

高原低氧对大鼠下丘脑谷氨酸、天门冬氨酸和NOS的影响

目的:观察高原低氧大鼠下丘脑谷氨酸(Glu)、天门冬氨酸(Asp)和一氧化氮合酶(NOS)的变化。方法:应用氨基酸测定和NADPH-d组化法,检测高原低氧模型大鼠下丘脑Glu、Asp含量和NADPH-d阳性神经元的数量。结果:高原低氧大鼠下丘脑Glu、Asp含量明显增多,室旁核、视上核可见密集深染的NADPH-d阳性神经元;用NMDA受体拮抗剂氯氨酮(Ketamine)和AP-V对高原低氧大鼠进行预处理后置于低压氧舱,观察到大鼠下丘脑室旁核、视上核NADPH-d阳性神经元数明显少于相应时间的高原低氧组(P＜0.01)。结论:NMDA受体可能参与了高原低氧引起的下丘脑NOS的表达。     

Protective effects of Big-leaf mulberry and physiological roles of nitric oxide synthases in the testis of mice following water immersion and restraint stress

Big-leaf mulberry is a new hybrid plant from the application of cell engineering technology, but its effect in stress-induced testicular dysfunction is unknown. Nitric oxide (NO) is a tiny, highly reactive lipophilic molecule produced by nitric oxide synthases (NOS). Three isoforms of NOS (neuronal NOS, inducible NOS and endothelial NOS) have been identified. Our aim was to investigate the effect of water immersion and restraint stress (WIRS) on NOS in the testis, and the effect of Big-leaf mulberry to protect against WIRS. The activity and expression of NOS, and total antioxidant capacity (T-AOC) in the mouse testis of different treatment groups (non-WIRS, 3 h-WIRS, WIRS-recovery) were examined. Histological analysis of WIRS-induced testicular damage and immunohistochemical staining of NOS were also analyzed. Results demonstrated that WIRS-exposed mice produced several injuries and showed an increased iNOS and eNOS mRNA expression in testes, whereas pretreatment with Big-leaf mulberry down-regulated iNOS and eNOS mRNA expressions and up-regulated T-AOC activities. Immunohistochemical studies showed that both iNOS and eNOS were localized in germ cells, spermatozoa and blood vessels in addition to Leydig cells and Sertoli cells, but nNOS was not present in these areas. In conclusion, our results suggested that Big-leaf mulberry exerted a protective effect on WIRS-induced testicular dysfunction, and iNOS and eNOS appeared to exert an important action in mouse testes exposed to WIRS.