浅蓝菌素对人结肠癌裸鼠移植瘤bcl-2及bax基因表达水平的影响

目的 :通过观察人结肠癌LoVo细胞裸鼠移植瘤bcl 2及bax基因表达水平 ,探讨脂肪酸合酶抑制剂浅蓝菌素选择性的抑瘤途径和机制。方法 :建立人结肠癌裸鼠移植瘤模型 ,浅蓝菌素每公斤体重 160mg腹腔注射 ,1次·d-1,连续10d ,第 17天处死并解剖动物 ,瘤组织作免疫组织化学检测bcl 2及bax基因表达水平。结果 :浅蓝菌素处理组结肠癌LoVo细胞移植瘤bcl 2基因蛋白表达率为 68.5 % ;对照组bcl 2蛋白阳性表达率为 87.8% ,阳性细胞弥漫分布。两组间有显著性差异 (P <0 .0 1)。bax基因蛋白在浅蓝菌素组移植瘤组织阳性表达率为 75 .4% ,主要呈强阳性表达 ;对照组阳性表达率为 9.5 %。两组间比较有统计学差异 (P <0 .0 1)。结论 :浅蓝菌素可使结肠癌LoVo细胞移植瘤的bcl 2基因表达下调 ,bax基因表达增强 ,提示bcl 2及bax基因在浅蓝菌素诱导的人结肠癌LoVo细胞凋亡中可能起着重要的调控作用。     

大鼠脑缺血再灌流脑区一氧化氮合酶的变化

研究大鼠脑缺血再灌注后大脑皮层,海马,纹状体和小脑组织一氧化氮合酶的变化。方法采用放射免疫法检测脑组织中ＮＯＳ的活性。结果大鼠脑缺血后５ｍｉｎ,各脑区结构型ＮＯＳ（ｃＮＯＳ）活性明显升高,持续到脑缺血３０ｍｉｎ,脑缺血３０－６０开始下降,诱导型ＮＯＳ（ｉＮＯＳ）活性在脑缺血后１０－１５ｍｉｎ开始升高,并持以脑缺血６０ｍｉｎ;脑缺血３０ｍｉｎ再灌注１５ｍｉｎ,各脑区ｃＮＯＳ和ｉＮＯＳ活性明显高于缺血     

诱导型一氧化氮合酶在实验性病毒性心肌炎心肌内的表达及其与心肌损害的关系

目的 探讨诱导型一氧化氮合酶（ｉＮＯＳ）在急性病毒性心肌炎心肌内的表达及其与心肌损害的关系。方法 ６～８周龄ＢＡＬＢ／Ｃ雄性小鼠２４只,随机分两组：感染组１６只以柯萨奇病毒Ｂ３ｍ（ＣＶＢ３ｍ）腹腔内注射,另８只为正常对照组。采用免疫组化法检测两组小鼠心肌组织内ｉＮＯＳ表达,并将其半定量与心肌病理变化程度进行相关分析,用比色法测定了血清中ＮＯ间接含量。结果 ①感染组小鼠心肌标本病理组织学检查均有不同     

间歇性气囊挤压大鼠腿部对挤压部位和远端骨骼肌一氧化氮合酶mRNA表达的影响

目的:为了进一步了解间歇性气囊挤压法(Intermittent pneumatic compression, IPC)挤压大鼠腿部与一氧化氮(NO)的关系.方法:检测了大鼠骨骼肌中3种一氧化氮合酶(NOS)同工酶:神经型NOS(nNOS);诱导型NOS(iNOS)和内皮细胞型NOS(eNOS)mRNA在IPC作用后的表达变化.25只SD大鼠被随机分为3个模拟实验组和4个IPC实验组.每只鼠取右侧胫前肌(AT)和提睾肌(CM)作为正常对照.IPC组挤压0.5,1,和5h,及挤压5h加等待4h,模拟实验组除不挤压外,其他操作均与实验组相同,然后分离左侧AT和CM作为处理后样品.所有样品应用RT-PCR进行NOS mRNA测定.以样品中看家基因2,3-二羟基丙醛-3-磷酸脱氢酶(GAPHD)cDNA为内参,与NOS cDNA 共同扩增.PCR产物电泳条带密度用NIH图像分析软件定量,并以与正常对照的对比值作为变化比率.结果:在IPC作用0.5、1和5h后,eNOS mRNA显著上升,在AT中分别达到正常对照的1.2,1.8和2.6倍;在CM中分别达到1.2,1.8和2.7倍,而其他NOS,除5hIPC组的nNOS外,总体表现下调.在IPC作用1h加等待4h组中,eNOS mRNA回复至正常对照水平.结论:该结果证实了IPC产生的机械压力至少部分增加了血管壁的剪切压,使内皮细胞增加了NO产物的释放量,导致了挤压部位及远端肌肉的血管扩张和改善了微循环.     

NMDA受体与NOS在大鼠脊髓中间外侧柱的定位和生后发育

目的 探讨N-甲基-天冬氨酸受体（NMDAR1和NMDAR2A/B亚基）、一氧化氮合酶（NOS）及还原型辅酶Ⅱ硫辛酰胺脱氢酶（NADPH-d）活性在大鼠脊髓中间外侧柱（IML）的定位与生后发育特征。方法 在甲醛固定的脊髓切片上,进行ABC法免疫染色和NADPH-d组织化学反应与半定量分析。结果 MMDAR1,NMDAR2A/B,NOS I和NADPH-d反应产物丰富地分布于IML,主要定位于神经元胞体、树突和轴突样纤维终末。在生后早期发育中它们具有明显的动态变化,生后7d(P7）有微弱或中等的表达,随后逐渐上调,P21或P28达到高峰,然后保持于此水平于成体动物。结论 NMDA受体-NO通路可能是脊髓交感节前神经元一条重要的细胞内信号途径,并参与神经元生后发育成熟的调控过程。     

诱导型一氧化氮合酶在哮喘大鼠肺组织的表达

目的　观察哮喘大鼠肺组织中诱导型一氧化氮合酶 (i NOS)活性的表达 ,探讨一氧化氮在哮喘大鼠气道炎症中的作用。方法　用卵白蛋白作为致敏原制备哮喘大鼠模型 ,用 SP免疫组化染色方法检测肺组织 i NOS的表达并观察 i NOS在气道组织分布的改变。结果　哮喘大鼠肺组织 i NOS的表达阳性率 (90 % )明显高于正常对照组 (2 0 % ) (P<0 .0 0 1)。哮喘大鼠气道组织 i NOS表达阳性细胞主要位于气道上皮细胞、气道平滑肌细胞、血管内皮和平滑肌细胞、浸润的中性粒细胞和巨噬细胞 ,而淋巴细胞表达不明显。用甲基强的松龙处理后哮喘大鼠肺组织i NOS表达阳性率 (30 % )明显降低。结论　以上结果提示一氧化氮在哮喘气道炎症中起重要作用 ,用甲基强的松龙治疗哮喘可以使哮喘大鼠肺组织中 i NOS表达阳性率降低 ,提示哮喘时产生过多的一氧化氮可能有加重气道炎症的作用     

骨肉瘤iNOS过度表达与血管生成及预后的关系

目的 探讨骨肉瘤中诱导型一氧化氮合酶（ｉＮＯＳ）的表达及其与血管生成和预后的关系。方法 应用免疫组化方法检测８０例骨肉瘤标本ｉＮＯＳ的表达,ＦⅧ－ＲＡ和ＣＤ３１免疫组化染色显示肿瘤内微血管密度。结果 ８０例骨肉瘤中５２例ｉＮＯＳ阳性（６５％）,肿瘤细胞境质中的巨噬细胞和部分血管内皮细胞均为阳性。ｉＮＯＳ阳性表达和肿瘤内微血管密度（ＭＶＤ）显著正相关,而且ｉＮＯＳ阳性组病人生成率显著低于ｉＮＯＳ阴性     

乙肝病毒标志物五种模式血清一氧化氮、一氧化氮合酶水平的比较

目的 :观察乙肝病毒 (HBV)感染者血清的一氧化氮 (NO)、一氧化氮合酶 (NOS)水平 ,探讨和研究NO在HBV感染过程中的变化和作用。方法 :应用ELISA法检测HBV血清标志物 ,并将HBV阳性血清分为五个常见模式组 ,应用Griess法测定各组血清NO和NOS水平与正常对照。结果 :HBsAg( +)、抗HBc( +)组 (第 4组 )和HBsAg( +)、HBeAg( +)、抗HBc( +)组 (第 5组 )显著降低 ;抗HBs( +)、抗HBc( +)组 (第 2组 )和HBsAg( +)、抗HBe( +)、抗HBc( +)组 (第 3组 ) ,NO和NOS回升 ;抗HBs( +)组 (第 1组 )和对照组水平相近。结论 :HBV在体内复制活跃时 ,可能抑制NOS活性 ,使NO合成减少 ,同时由于NO发挥对肝细胞的保护作用而使消耗增加。检测血清NO、NOS可观察HBV感染程度和预后。NO用于HBV感染的治疗有待研究     

Effects of ibuprofen enantiomers and its coenzyme A thioesters on human prostaglandin endoperoxide synthases

1. Ibuprofen enantiomers and their respective coenzyme A thioesters were tested in human platelets and blood monocytes to determine their selectivity and potency as inhibitors of cyclo-oxygenase activity of prostaglandin endoperoxide synthase-1 (PGHS-1) and PGHS-2.
2. Human blood from volunteers was drawn and allowed to clot at 37°C for 1 h in the presence of increasing concentrations of the test compounds (R-ibuprofen, S-ibuprofen, R-ibuprofenoyl-CoA, S-ibuprofenoyl-CoA, NS-398). Immunoreactive (ir) thromboxane B₂ (TXB₂) concentrations in serum were determined by a specific EIA assay as an index of the cyclo-oxygenase activity of platelet PGHS-1.
3. Heparin-treated blood from the same donors was incubated at 37°C for 24 h with the same concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 μg ml⁻¹). The contribution of PGHS-1 was suppressed by pretreatment of the volunteers with aspirin (500 mg; 48 h before venepuncture). As a measure of LPS induced PGHS-2 activity immunoreactive prostaglandin E₂ (irPGE₂) plasma concentrations were determined by a specific EIA assay.
4. S-ibuprofen inhibited the activity of PGHS-1 (IC₅₀ 2.1 μM) and PGHS-2 (IC₅₀ 1.6 μM) equally. R-ibuprofen inhibited PGHS-1 (IC₅₀ 34.9) less potently than S-ibuprofen and showed no inhibition of PGHS-2 up to 250 μM. By contrast R-ibuprofenoyl-CoA thioester inhibited PGE₂ production from LPS-stimulated monocytes almost two orders of magnitude more potently than the generation of TXB₂ (IC₅₀ 5.6 vs 219 μM).
5. Western blotting of PGHS-2 after LPS induction of blood monocytes showed a concentration-dependent inhibition of PGHS-2 protein expression by ibuprofenoyl-CoA thioesters.
6. These data confirm that S-ibuprofen represents the active entity in the racemate with respect to cyclo-oxygenase activity. More importantly the data suggest a contribution of the R-enantiomer to therapeutic effects not only by chiral inversion to S-ibuprofen but also via inhibition of induction of PGHS-2 mediated by R-ibuprofenoyl-CoA thioester.
7. The data may explain why racemic ibuprofen is ranked as one of the safest non-steroidal anti-inflammatory drugs (NSAIDs) so far determined in epidemiological studies.

     

Nitric oxide synthase activity is inducible in rat,but not rabbit alveolar macrophages,with a concomitant reduction in arginase activity

Alveolar macrophages were obtained by broncho-alveolar lavage of isolated rat and rabbit lungs and cultured (2.5 × 10⁶ cells/dish) for 18 h in the absence or presence of bacterial lipopolysaccharides (LPS) alone or in combination with cytokines. Thereafter, accumulation of ³H-citrulline (NO synthase activity) and ³H-ornithine (arginase activity) were determined.During incubation of rat alveolar macrophages with ³H-arginine clear amounts of ³H-citrulline and ³H-ornithine (3.8 and 4.6% of the added ³H-arginine, respectively) were formed and most of these metabolites appeared in the incubation medium (ratios extra-/intracellular of 17 and 70 for ³H-citrulline and ³H-ornithine, respectively). When rat alveolar macrophages had been cultured with LPS the formation of ³H-citrulline was increased about 30-fold and this was accompanied by a reduction in ³H-ornithine formation of about 60%. The effects of LPS were largely attenuated by dexamethasone (10 mol/1). Inhibition of NO synthase by N^G-monomethyl-l,-arginine (l-NMMA, 100 mol/1) in LPS treated alveolar macrophages reduced the formation ³H-citrulline by more than 90% and restored the ³H-ornithine formation. After culturing in the presence of LPS the ratios extra/intracellular of ³H-citrulline and ³H-ornithine were markedly enhanced and this effect was not dexamethasone sensitive. During incubation of rabbit alveolar macrophages a marked formation of ³H-ornithine (about 5.3% of the added ³H-arginine), but no significant formation of ³H-citrulline could be detected. Pretreatment with LPS tended to enhance the formation of ³H-ornithine (by 50%) without effects on ³H-citrulline. Rabbit-interferon and/or tumor necrosis factor- present together with LPS during the culture period did not result in a significant ³H-citrulline formation. Under all conditions tested, culture media of rabbit alveolar macrophages did not contain significant amounts of nitrite (less than 0.5 nmol) whereas in culture media of untreated rat alveolar macrophages 22 nmol nitrite (per 18 h) were detected, and LPS induced a 3-fold nitrite accumulation, an effect prevented by dexamethasone.In conclusion, in rabbit alveolar macrophages NO synthase activity was not detectable and could also not be induced by LPS and different cytokines, whereas in rat alveolar macrophages NO synthase was readily inducible. Alveolar macrophages of both species showed marked arginase activity. After induction of marked NO synthase activity, ornithine formation was largely reduced possibly by concomitant inhibition of arginase and/or withdrawn of arginine from arginase.