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。     

糖尿病大鼠睾丸病理变化及脂质过氧化和一氧化氮的改变

目的:研究糖尿病睾丸的病理变化及其发生机制。方法:用光镜及透射电镜观察四氧嘧啶诱导的糖尿病1个月大鼠睾丸的形态学改变,并测定睾丸组织超氧化物歧化酶(SOD)、谷胱甘肽过氧化物酶(GSH-PX)、一氧化氮合酶(NOS)的活性及一氧化氮(NO)、丙二醛(MDA)含量。结果:光镜下主要表现为睾丸曲细精管萎缩及生精阻滞;透射电镜下主要见到支持细胞线粒体与内质网扩张、胞浆内内含物形成;睾丸组织的SOD、GSH-PX活性实验组低于对照组,NOS活性及NO、MDA含量实验组高于对照组。结论:糖尿病大鼠睾丸病变主要为支持细胞受损及生精障碍,其可能与脂质过氧化作用及NO所致的损伤有关。     

一氧化氮对慢性低氧大鼠肺循环组织细胞增殖/凋亡的影响

目的：探讨一氧化氮（NO）对低氧条件下细胞增殖/凋亡的影响，了解NO在肺循环结构改建的作用。方法：复制间断常压缺氧大鼠模型，N^ω-硝基L-精氨酸甲酯（L-NAME）和5-单硝酸异山梨酯干预内外源NO。测定mPAP、R/L+S、血NO、MDA和SOD水平，对肺、心肌、肺动脉组织作PCNA免疫组化染色和细胞凋亡原位缺口末端DNA碎片标记。结果：①HY和HY+NAME组大鼠mPAP高于NS组，NO和SOD水平低于NS组（P<0.01）；HY+IS组mPAP明显低于HY组（P<0.01）。②HY组大鼠肺组织、心肌、肺动脉壁细胞增殖指数（PI）明显高于NS组（P<0.01）；细胞凋亡指数（AI）无明显差异。③HY+NAME组大鼠肺、心肌、肺动脉PI/AI比值均显著高于NS组（P<0.01），而HY+IS组大鼠上述组织PI/AI比值明显低于HY组（P<0.05）。结论：①NO参与慢性低氧性PH的发生和发展。②慢性低氧性PH存在细胞增殖/凋亡失衡，细胞增殖大于凋亡，造成肺循环结构改建。③NO可通过诱导细胞凋亡来减轻肺循环结构重建。     

Determination of nitric oxide synthase activity and apoptosis of germ cells in different obstruction models

We aimed to determine the changes of inducible nitric oxide synthase (iNOS) and endothelial nitric oxide synthase (eNOS) immunoreactivity and apoptosis after proximal and distal obstruction models on ipsilateral and contralateral testicular tissues. Male albino Wistar rats were randomly divided into three groups (n=30): a control group which underwent sham operations (n=10), a unilateral vasal ligation (n=10) and a unilateral epididymal ligation group (n=10). iNOS and eNOS distribution and apoptosis were studied in both ipsilateral and contralateral testes using quantitative immunohistochemistry. Nitric oxide synthase activity was significantly affected in ipsilateral and contralateral testes cells after vasal and epididymal ligation. eNOS immunoreactivity increased markedly after ipsilateral vasal ligation (ILVL). Degeneration-related changes were also associated with changes in apoptotic rate. Analysis using the terminal dUTP nick end-labeling TUNEL method revealed that apoptotic cell numbers significantly increased after ILVL. p53 and bcl-2 immunoreactivity increased in both experimental groups compared with the sham-operated group. Changes in iNOS and eNOS immunolocalisation were strongly associated with cell damage, because germ cell degeneration was more prominent in the ILVL group. Altered p53 immunolocalisation was also associated with cell degeneration, and a rise in bcl-2 immunoreactivity might be considered to reflect a protective mechanism in the testis. These cellular changes could enlighten understanding of the interaction between testicular functioning and damage.     

The role of ATP sensitive K+ channels and of nitric oxide synthase on myocardial ischemia/reperfusion-induced apoptosis

During ischemia, ATP-sensitive K+ channels (KATP channels) open, and this triggers necrotic processes and apoptosis. In this study, we investigated whether selective sarcoplasmic and mitochondrial KATP channel blockers affected myocardial apoptosis and nitric oxide synthase (NOS) activity in a rat model of myocardial ischemia/reperfusion in vitro. Isolated rat hearts were subjected to 30 min of coronary artery occlusion followed by 30 min of reperfusion. A selective sarcKATP channel blocker, HMR1098 and a selective mitoKATP channel blocker, 5-hydroxydecanoate, were added to the perfusion fluid 10 min before occlusion. Myocardial apoptosis was detected immunohistochemically using the TUNEL method. Myocardial inducible NOS (iNOS) and endothelial NOS (eNOS) were determined immunohistochemically. In control hearts, apoptosis induction was associated with a greater immunoreactivity of iNOS than eNOS. Treatment with HMR1098, at a concentration of 3 micromol/l, significantly reduced the TUNEL-positive cardiomyocytes and this was associated with decreased iNOS and increased eNOS immunoreactivity. When this drug was administered at a higher concentration, at 30 micromol/l, a more marked reduction in apoptosis was observed but, in contrast to the effects observed at the lower drug concentration, eNOS immunoreactivity was almost completely abolished while iNOS was strong. Moreover, ischemia-induced cardiac dysfunction (e.g. contractile force and recovery of coronary flow) was increased by the higher concentration of HMR 1098. In hearts treated with 5-hydroxydecanoate, myocyte apoptosis was slightly reduced, and this was associated with an almost equal increase in both iNOS and eNOS immunoreactivity. These findings suggest that iNOS appears to be more important than eNOS in the reduction of apoptosis. However, the further inhibition of apoptosis by the higher concentration of HMR 1098 was associated with poorer cardiac function.     

一氧化氮和caspase-3在多巴胺诱导PC12细胞

目的：探讨一氧化氮（NO）和半胱氨酸蛋白酶3（caspase-3）在多巴胺诱导PC12细胞凋亡中的可能作用。方法：流式细胞仪定量检测PC12细胞的凋亡率,原位末端标记法（TUNEL）观察凋亡细胞的形态,Griess法测定NO₂^-的浓度,荧光分光光度计法检测caspase-3的活力,半定量RT-PCR法检测诱导型一氧化氮合酶（iNOS）mRNA的表达水平。结果：多巴胺（0.15-0.60 mmol/L）可剂量依赖性地诱导PC12细胞凋亡,表现为凋亡细胞的TUNEL染色阳性;iNOS mRNA的表达、NO的合成及caspase-3的活力均有明显的增加（P<0.01）;特异性的iNOS抑制剂aminoguanidine和caspase-3抑制剂Ac-DEVD-CHO通过减少NO的生成和抑制caspase-3的激活阻断PC12细胞凋亡。结论：NO的生成可能是多巴胺诱导PC12细胞凋亡的触发因子,caspase-3的激活是其中的效应因子。     

葛根素对糖尿病大鼠睾丸的保护作用

目的： 探讨葛根素对糖尿病大鼠睾丸的保护作用及其机制。方法：雄性SD大鼠随机分成6组：正常对照组,糖尿病模型组,葛根素高(160 mg·kg-1)、中(120 mg·kg-1)、低(80 mg·kg-1)剂量治疗组,氨基胍(100 mg·kg-1)治疗组;各组大鼠每天腹腔注射相应药物1次,正常对照组及糖尿病模型组腹腔注射等体积丙二醇。治疗12周后,透射电镜下观察睾丸的形态学改变,用生化、免疫学方法测定睾丸组织中一氧化氮（NO）、内皮素-1（ET-1）、降钙素基因相关肽（CGRP）、丙二醛（MDA)的含量及一氧化氮合酶（NOS）、诱导型NOS(iNOS)、超氧化物歧化酶(SOD)、Ca2+-ATPase、Na+-K+-ATPase的活性,采用RT-PCR检测睾丸组织内皮型NOS(eNOS)、iNOS、CGRP、ET-1、醛糖还原酶（AR）的基因表达水平。结果：糖尿病组大鼠睾丸组织电镜下主要见到支持细胞与曲细精管基底膜接触面减小、出现空泡,胞浆内内含物形成;NO、CGRP含量及SOD、Ca2+-ATPase、Na+-K+-ATPase活性和eNOS、CGRP mRNA表达明显低于正常对照组（P<0.05或P<0.01）,而ET-1、MDA含量及NOS、iNOS活性和iNOS、AR、ET-1 mRNA表达明显高于正常对照组（均P<0.01）。经葛根素治疗后,上述改变逆转,与糖尿病模型组比较差异显著（P<0.05或P<0.01）,电镜下睾丸组织的形态结构接近于正常组;而葛根素高、中、低剂量治疗组、氨基胍治疗组间差异无显著。结论：葛根素对糖尿病大鼠睾丸具有一定的保护作用,其机制可能与改善睾丸血供及减轻氧化应激损伤有关。     

脑挫伤后一氧化氮合酶阳性细胞及一氧化氮含量的变化

目的　探讨脑挫伤后一氧化氮合酶 (NOS)阳性细胞和一氧化氮 (NO)的变化和意义。方法　采用自由落体法致Wistar大鼠顶叶皮质挫裂伤动物模型。伤后 2 4h、72h和 7d取脑 ,制作冰冻切片 ,采用NADPH组织化学染色 ,显示脑挫伤区NOS阳性细胞。用硝酸还原酶法测定血液和脑组织中NO含量。结果　脑挫伤后 72h ,NOS阳性细胞数密度 (Nv)和面密度(Sv)明显增高 (P <0 0 5 ) ,而且 7d时仍无明显下降。血液和脑中NO含量也增高 ,并与NOS细胞呈平行关系。结论　脑挫伤后不同时间NOS细胞数目和NO含量有明显改变 ,提示NOS和NO参与了脑挫伤的病理过程     

肢体缺血再灌注后的肺损伤和细胞凋亡及NO的效应

目的：探讨肢体缺血再灌注（LIR）后肺的损伤性变化以及细胞凋亡在肺损伤发生中的作用；探讨一氧化氮（NO）对LIR后肺组织细胞凋亡的影响。 方法：采用本室常规方法复制大鼠LIR模型，给予外源性一氧化氮合酶底物（L-Arg）和一氧化氮合酶抑制剂(L-NAME)处理，采用原位末端标记法（TUNEL）检测缺血4 h再灌注4 h时各组动物肺组织细胞凋亡情况；采用放免法检测凋亡相关细胞因子TNF-α在肺组织的表达，结合计算机分析系统对结果进行定量分析；采用免疫组织化学方法检测Bcl-2、Bax、caspase-3、TNF-α蛋白表达情况，结合自动图像分析系统对其结果进行定量分析；在光镜下观察肺组织的形态学改变。结果：大鼠LIR后4 h，肺泡Ⅱ型上皮细胞、肺血管内皮细胞呈凋亡改变,肺组织TNF-α、caspase-3、Bax明显上调,Bcl-2表达下调。L-Arg处理组，凋亡细胞数明显减少，肺组织TNF-α、caspase-3、Bax的表达情况与IR组相比明显减弱，Bcl-2表达明显增强；L-NAME处理组动物肺组织TNF-α、caspase-3、Bax的表达情况与IR组相比明显增强，Bcl-2表达明显减弱。结论：细胞凋亡参与了大鼠LIR后急性肺损伤的发生，且与TNF-α有关；NO可通过减弱细胞凋亡相关因子TNF-α的表达，减轻LIR后肺组织的细胞凋亡。     

一氧化氮/诱导型一氧化氮合酶在动脉粥样硬化过程中的作用

 目的：探讨一氧化氮（NO）/诱导型一氧化氮合酶（iNOS）在动脉粥样硬化（atherosclerosis,AS）过程中的动态变化,分析其对动脉粥样硬化形成过程的影响。方法：将60只SD大鼠随机分成2组：对照组及AS组,每组30只。AS组采用维生素D3腹腔注射联合高脂饲料饲养的方法构建动脉粥样硬化模型。用相关生化方法检测血清各项生化指标：总胆固醇、甘油三酯、高密度脂蛋白胆固醇、低密度脂蛋白胆固醇、空腹血糖和钙离子,比色法检测血清NO浓度,并对主动脉行HE染色,免疫组化技术检测iNOS蛋白表达,将所得数据进行统计分析,用简单线性相关分析NO与钙离子及动脉粥样硬化指数的相关性。结果：90 d后成功构建了主动脉中膜钙化型动脉粥样硬化模型。血清NO浓度在动脉粥样硬化过程中逐步下降,各组间差异均有统计学意义（均P<0.05）。动脉粥样硬化过程中动脉粥样硬化指数与钙离子呈正相关,与NO呈负相关。在90 d的AS组粥样斑块区免疫组化技术检测到iNOS蛋白表达。结论:在动脉粥样硬化形成过程中,主动脉粥样斑块区iNOS蛋白高表达,但血清NO浓度逐渐降低,NO抗动脉粥样硬化作用减弱。     

The role of mitochondria in nitric oxide-mediated thymocyte apoptosis

The aim of this study was to analyze whether immunosuppressants could affect NO-induced thymocytes apoptosis. S-Nitroso-N-acetyl-D,L-penicillamine (SNAP, an NO donor) has an apoptotic effect on murine thymocytes. This NO-induced thymocyte apoptosis is associated with a reduced uptake of DiOC(6), a fluorochrome which incorporates into cells depending on their mitochondrial membrane potential (DeltaPsi(m)). The immunosuppressant, cyclosporin A, partially inhibited NO-induced thymocyte apoptosis as well as the reduction of DeltaPsi(m). In contrast, another immunosuppressant, FK506, did not affect NO-induced thymocyte apoptosis. Although FK506 and cyclosporin A inhibit T-cell signal transduction by the same mechanism, their effects on mitochondria may contribute to the differential apoptosis induction ability.     

一氧化氮诱导大鼠肺动脉平滑肌细胞凋亡机制研究

目的:研究一氧化氮(NO)诱导大鼠肺动脉平滑肌细胞(PASMC)凋亡的作用机制。方法:体外培养Wistar大鼠PASMC,加入NO供体硝普钠(SNP)于常氧和低氧条件下孵育12h或24h,通过流式细胞术碘化丙啶染色法观察PASMC细胞周期时段及凋亡亚二倍体峰变化,应用细胞免疫化学法检测PASMCcaspase-3和NF-κB的表达,同时行DNA断裂琼脂糖凝胶电泳。结果:SNP作用后PASMC出现剂量-依赖性促凋亡作用(P＜0.01),表现为凋亡指数与caspase-3表达的不同程度的增强。随凋亡的进展,出现PASMC凋亡核小体DNAladder,同时PASMCNF-κB阳性核表达较对照组少(P＜0.01)。结论:外源性NO诱导大鼠PASMC凋亡,caspase-3与NF-κB可能涉及PASMC凋亡的调控信号途径。     

肝移植围术期血浆一氧化氮和一氧化氮合酶活性的变化及意义

目的：动态观察肝移植围术期血浆一氧化氮（NO）水平和一氧化氮合酶（NOS）活性的变化，并探讨其意义。 方法： 30例终末期肝病患者接受原位肝移植术。用放免法、比色法分别测定肝移植围术期5个时点血浆NO2-/NO3-水平和NOS活性，观察其动态变化。同步抽取桡动脉和肺动脉血做血气分析，记录不同时期的PO2、PCO2、SO2、Hb，根据肺内分流标准模型公式计算(Qs/Qt)。并监测围术期心输出量（CO）、心率（HR）、中心静脉压（CVP）、平均动脉压（MABP）、体循环阻力（SVR）。 结果： (1) 无肝前10 min NO2-/NO3-水平明显高于麻醉后术前。无肝期30 min NO2-/NO3-显著低于无肝前10 min。新肝期30 min NO2-/NO3-显著高于麻醉后术前、无肝期30 min。（2）TNOS活性各时点无显著差异。无肝前10 min、新肝30 min时iNOS活性明显高于麻醉后术前。与无肝30 min值比较，新肝期30 min iNOS活性显著升高。（3）MABP在开放下腔静脉后1 min明显下降，CO和CVP在无肝期下降，新肝期增高。SVR在无肝期增高，新肝期明显下降。（4）Qs/Qt在无肝期下降，新肝期30 min升高。 结论： 在肝移植围术期各个时段，NO水平及iNOS活性各不相同。高NO水平可能是新肝期低阻力、肺内分流增加的原因。     

实验性精索静脉曲张大鼠睾丸组织和血清一氧化氮和一氧化氮合酶的改变

目的：检测实验性精索静脉曲张大鼠睾丸组织和血清NO含量和NOS活性的改变，以进一步阐明精索静脉曲张的发生机制。方法：通过手术方法建立大鼠精索静脉曲张模型，检测NO和NOS的变化。结果：实验组左侧睾丸组织NO含量显著高于对照组左侧睾丸含量；实验组血清NOS活力明显低于对照组。结论：实验性精索静脉曲张大鼠睾丸组织和血清NO和NOS有改变。     

Epigallocatechin gallate inhibits nitric oxide-induced apoptosis in rat PC12 cells

Nitric oxide (NO) is associated with many pathophysiology of the central nervous system including brain ischemia, neurodegeneration and inflammation. Epigallocatechin gallate (EGCG) is a major compound of green tea polyphenol that has shown the protective activity against neuronal diseases. This study examined the effect of EGCG on NO-induced cell death in PC12 cells. The administration of sodium nitroprusside (SNP), a NO donor, decreased the cell viability and induced apoptosis showing characterization such as cell shrinkage and chromatin condensation as well as subG1 fraction of cell cycles. EGCG inhibited the cytotoxicity and apoptotic morphogenic changes induced by SNP. EGCG attenuated the production of reactive oxygen species (ROS) by SNP, and ameliorated the SNP-induced Bax to Bcl-2 expression ratio leading to apoptosis. In addition, EGCG prevented the release of cytochrome c from the mitochondria into the cytosol as well as the upregulation of the voltage-dependent anion channel (VDAC), a cytochrome c releasing channel, in the mitochondria of SNP-treated cells. EGCG abrogated the activation of caspase-9, caspase-8 and caspase-3 induced by SNP. These results demonstrate that EGCG has a protective effect against SNP-induced apoptosis in PC12 cells by scavenging ROS and modulating the signal molecules associated with cytochrome c, caspases, VDAC and the Bcl-2 family. These findings suggest that EGCG might be a natural neuroprotective substance.     

一氧化氮对大鼠内毒素血症肠道的保护作用及机理研究

本文用一氧化氮（ＮＯ）合酶抑制剂硝基左旋精酸（Ｌ－ＮＮＡ）及其底物左旋精氨酸（Ｌ－Ａｒｇ）对比观察了两者对内毒素血症大鼠肠道损伤的影响，并从白细胞，自由基及血流改变等方面探讨了其作用机理。结果表明：用Ｌ－ＮＮＡ（４ｍｇ／ｋｇ）抑制ＮＯ合成可加重内毒素（１０ｍｇ／ｋｇ）引起的肠微血管和粘膜损伤，表现为肠组织伊文氏蓝含量增加，二胺氧化酶活性降低，同时肠组织髓过氧化物酶及丙二醛含量明显升高，肠血流降低：     

大鼠肺纤维化形成中内源性一氧化氮对肺泡上皮细胞凋亡的影响

目的:研究大鼠肺纤维化形成过程中异常增多的肺内源性一氧化氮对肺泡上皮细胞凋亡的影响。方法:气管内滴注博莱霉素,用硝酸还原法检测出肺血NO₂^-/NO₃^-含量;TdT介导的dUTP缺口末端标记法(TUNEL)和透射电镜观察肺泡上皮细胞凋亡。分别观察注BLMA₅后14 d和30 d以及用氨基胍(AG)阻断iNOS合成NO后上述指标的变化。结果:(1)注BLMA₅后14 d,出肺血NO₂^-/NO₃^-含量分别高于正常对照组和注BLMA₅后30 d组(均P<0.01);凋亡的肺泡上皮细胞数亦分别多于正常对照和注BLMA₅后30 d组(均P<0.01)。(2)AG阻止出肺血NO₂^-/NO₃^-含量增高的同时,亦抑制了肺泡上皮细胞的凋亡。结论:在肺纤维化形成过程中,内源性NO的增多,有诱导肺泡上皮细胞凋亡的作用。     

The expression and activity of renal nitric oxide synthase and circulating nitric oxide in polycystic kidney disease rats

Nitric oxide (NO) influences tubular fluid and electrolyte transport, and hence possibly also fluid accumulation in renal cysts. The expression and activity of intrarenal constitutive NO synthase (cNOS) [neuronal NOS, nNOS and endothelial NOS, eNOS] and inducible NOS (iNOS) and plasma nitrite/nitrate (PNOx) concentration were assessed in homozygous Han:SPRD polycystic kidney disease (PKD) rats (cy/cy), heterozygous Han:SPRD PKD rats (cy/+), homozygous normal Han:SPRD littermates (+/+) and Sprague Dawley rats (sd). The results showed: 1) nNOS expression was decreased in proximal tubules and thick ascending limbs of the loop of Henle in cy/cy and cy/+ rats compared to +/+ and sd rats (p<0.05). nNOS was weakly expressed in the epithelium of small cysts and unexpressed in epithelium of large cysts. 2) iNOS expression was increased in proximal tubular epithelial cells in cy/+ rats compared to +/+ rats and sd rats (p<0.01). iNOS expression in cyst epithelium was decreased in cy/+ rats (p<0.05) and absent in cy/cy rats. 3) eNOS expression was similar in the endothelium of intrarenal arteries in all groups. 4) The activity of renal cNOS was decreased in cy/cy and cy/+ rats; the activity of iNOS was decreased only in cy/cy rats, with no significant difference among the other three groups. 5) PNOx concentration was higher in cy/cy rats than in the other three groups, and correlated positively with plasma creatinine and urea. In conclusion, NOS expression and activity decreased as cysts developed, suggesting that NO downregulation is involved in the pathogenesis of PKD.     

环磷酰胺对大鼠睾丸和附睾的影响

目的探讨抗肿瘤药物所致大鼠睾丸和附睾损害,为寻找男性不育症中药治疗的研究提供理论依据。方法选用16只15周龄SD大鼠随机分为对照组和实验组,每组8只;实验组腹腔注射环磷酰胺20mg·kg-1·d-1,连续5d,用药2个月后,应用HE染色法研究大鼠睾丸、附睾远期组织学变化,用原位缺口末端标记法(TUNEL方法)检测生精细胞凋亡。结果实验组大鼠体重、睾丸和附睾重量均显著减轻(P<0.01),睾丸生精小管直径缩小、间距增宽、生精上皮变薄、生精细胞层次和数量减少、生精小管腔多未见精子形成,实验组睾丸生精小管直径、面积、生精上皮细胞数、均显著低于对照组(P<0.01);实验组生精细胞凋亡增多,与对照组比较,生精细胞显著凋亡(P<0.01);附睾管管腔内腔内精子稀少,含有大量脱落细胞,管壁变薄。结论环磷酰胺对大鼠睾丸、附睾远期损害明显,促进生精细胞凋亡。