瘦素对RAW264.7细胞肿瘤坏死因子α表达的影响

背景:瘦素是脂肪组织分泌的一种多肽激素,研究显示瘦素在动脉粥样硬化形成中发挥了一定重要的作用。目的:观察瘦素对鼠源性巨噬细胞系RAW264.7细胞肿瘤坏死因子α表达的影响,并从核转录因子κB活性变化角度探讨其可能机制。设计:对照观察实验。单位:华中科技大学同济医学院生物化学及分子生物学系。材料:实验于2005-04/2006-02在华中科技大学同济医学院生物化学及分子生物学系及附属协和医院普外科实验室完成。将培养的RAW264.7细胞分为不同浓度瘦素处理组(12.5,25,50,100μg/L)、IkappaB激酶抑制剂组及空白对照组。每组3瓶,重复实验3次。方法:将鼠源性巨噬细胞株RAW264.7细胞以1×109L-1密度接种于6孔板中,用含体积分数为0.1的小牛血清的RPMI-1640培养基培养。待RAW264.7细胞生长至80%时,换用无血清培养基Opti-MEM继续培养24h后,将细胞分为不同浓度瘦素处理组(12.5,25,50,100μg/L)及空白对照组,瘦素孵育4h后采用反转录-聚合酶链反应检测肿瘤坏死因子α在mRNA水平的表达。上述分组细胞经瘦素分别孵育1,3,6和9h后采用双抗夹心酶联免疫吸附实验检测肿瘤坏死因子α在蛋白水平的表达。上述分组细胞经瘦素孵育不同时间后采用凝胶迁移率实验检测细胞核内核转录因子κB活性。将RAW264.7细胞分为以下4组:空白对照组、IkappaB激酶特异性抑制剂PS1145(10μmol/L)处理组、瘦素(50μg/L)处理组、瘦素(50μg/L) PS1145(10μmol/L)组,各组孵育时间均为6h,分别检测细胞核内核转录因子κB活性及肿瘤坏死因子α在mRNA水平的表达。主要观察指标:①不同浓度瘦素对RAW264.7细胞肿瘤坏死因子α:mRNA表达水平的影响;蛋白分泌的影响。②不同浓度瘦素对RAW264.7细胞核内核转录因子κB活性的影响。③抑制IkappaB激酶活性对瘦素诱导RAW264.7细胞肿瘤坏死因子α的影响。结果:①RAW264.7细胞经不同浓度的瘦素处理后,肿瘤坏死因子α在mRNA水平呈瘦素剂量依赖性增加,50μg/L瘦素处理组达峰值。②蛋白水平的表达呈瘦素剂量时间依赖性增加,50μg/L瘦素处理6h即可达峰值。③核转录因子κB的活性亦与瘦素浓度正相关,50μg/L瘦素处理6h后核转录因子κB活性最高(P<0.05)。④抑制IkappaB激酶活性可部分抑制肿瘤坏死因子α的表达。结论:瘦素可直接促进RAW264.7细胞肿瘤坏死因子α的表达和分泌,并呈剂量时间依赖性,其机制可能与瘦素激活核转录因子κB有关。这可能是瘦素致动脉粥样硬化的机制之一。     

血清瘦素、肿瘤坏死因子与儿童厌食症的相关性研究

儿童厌食症是指较长期的食欲减退或消失,儿童厌食的发病率约12．0％～34．0％,且近年来呈上升趋势,长期厌食严重影响小儿的生长发育、营养状况以及智力发育。瘦素是脂肪细胞分泌的一种蛋白激素．具有广泛的生理作用,它对食欲及能量代谢也具有重要的调节作用．肿瘤坏死因子（TNF-α）的氨基酸序列与恶液质素十分相近,被认为是同一种蛋白质,它们产生食欲不振的机制可能是作用于下丘脑或抑制胃排空。     

胃肠道肿瘤患者血清瘦素和肿瘤坏死因子水平与营养状态的关系

目的研究胃肠道恶性肿瘤患者血清瘦素和肿瘤坏死因子(TNF-α)的变化,以及其与营养状态的关系。方法采用放射免疫法测定63例胃肠道恶性肿瘤患者和50例正常人的血清瘦素及TNF-α水平,并分析血清瘦素和TNF-α水平与体质量指数(BMI)、三头肌皮皱厚度(TSF)、上臂中部臂围(MAC)和血清白蛋白(ALB)等营养参数的关系。结果胃肠道恶性肿瘤患者的血清瘦素水平下降,TNF-α浓度升高,与对照组比较有显著性差异(P<0.01);血清瘦素水平与BMI、TSF、MAC呈明显正相关,与TNF-α无相关性(P>0.05)。结论胃肠道恶性肿瘤患者的血清瘦素水平下降,TNF-α水平升高,血清低瘦素水平降低对食欲的刺激,可能参与胃肠道恶性肿瘤恶液质的发生。     

急性心肌缺血损伤中过氧化物酶体增殖物激活受体α活化与肿瘤坏死因子仪表达的关系

目的：观察预先应用过氧化物酶体增殖物激活受体α的激动剂非诺贝特活化过氧化物酶体增殖物激活受体α，对急性心肌缺血性损伤大鼠血清肿瘤坏死因子α的影响。方法：实验于2004—06／2005—05在哈尔滨医科大学克山病研究所完成。取30只健康纯系雄性Wistar大鼠随机分为异阿肾上腺索组、非诺贝特组和正常对照组3组，每组10只。①非诺贝特溶于30g/L阿拉伯树胶中制成混悬液．非诺贝特组给予非诺贝特80mg／kg灌胃，其他两组给予等量的30g/L阿拉伯树胶灌胃，1次／d，共7d。②末次灌胃后1h异丙肾上腺素组和非诺贝特组给予异丙肾上腺素5mg／kg腹腔注射复制急性心肌缺血损伤模型；正常对照组给予等量的生理盐水腹腔注射。异丙肾上腺素注射24h，麻醉后取血清和部分心脏，用酶联免疫吸附实验测定血清中肿瘤坏死因子α的浓度，用反转录-聚合酶链反应测定心肌组织中过氧化物酶体增殖物激活受体αmRNA表达水平。结果：30只大鼠进入结果分析。（1）血清中肿瘤坏死因子α的浓度：异丙肾上腺素组和非诺贝特组高于正常对照组[（301．72&;#177;2．49），（164．29&;#177;3．60），（16．35&;#177;2．37）ng／L，P〈0．01]，但非诺贝特组低于异丙肾上腺素组（P〈0．01）。②心肌组织中过氧化物酶体增殖物激活受体αmRNA表达水平：异丙肾上腺素组和非诺贝特组低于正常对照组（0．7355&;#177;0．0455，0．8863&;#177;0．0322，0．9267&;#177;0．03l9，P〈0．01）；但非诺贝特组高于异丙肾上腺素组（p〈0．01）。结论：过氧化物酶体增殖物激活受体α活化后参与急性心肌缺血性损伤中的炎症反应，显著抑制肿瘤坏死因子α表达。提示非诺贝特对心肌缺血有一定保护作用。     

肿瘤坏死因子α对体外血脑屏障模型通透性的影响

背景:以往的研究发现,感染性脑损伤时脑组织中白细胞介素1和肿瘤坏死因子α含量明显增加,并与脑损害呈正相关关系,其能否导致血脑屏障通透性增高?目的:观察肿瘤坏死因子α对体外血脑屏障模型通透性的影响及其可能的调控机制。设计:体外细胞模型对照实验。单位:中南大学湘雅医院儿科,中南大学湘雅医学院生化系。材料:选用20只生后7d健康SD大鼠,雌雄不拘,清洁级,由中南大学湘雅医学院动物部提供;肿瘤坏死因子α购自sigma公司;DMEM液体培养基、胎牛血清购自Hyclone;Rhok的特异性拮抗剂Y-27632购自Alexis公司,兔抗人Ⅷ因子相关抗原购自Zymed公司;小鼠抗大鼠神经胶质纤维酸性蛋白单抗(GFAP)购自Neomarkers公司。方法:实验于2004-03/2005-04在中南大学湘雅医院完成。利用脑微血管内皮细胞与星型胶质细胞共培养建立体外大鼠血脑屏障模型,共培养至10d开始干预,实验分为模型组、Y-27632对照组、肿瘤坏死因子α干预组和Y-27632预处理组。模型组仅给予血脑屏障模型制备,肿瘤坏死因子α干预组向血脑屏障模型内加入肿瘤坏死因子α0.01g/L干预5h;Y-27632预处理组指向血脑屏障模型内加入Y-2763230μmol/L预处理1h后,再予肿瘤坏死因子α0.01g/L干预5h,Y-27632对照组造模后仅加入Y-27632干预,方式同Y-27632预处理组。取分组处理后待用模型,分别于30,60,120,240min采用γ计数仪检测125Ⅰ-牛血清白蛋白的通透量观察肿瘤坏死因子α对血脑屏障通透性的影响。主要观察指标:各组干预后不同时间点体外大鼠血脑屏障模型对125Ⅰ-牛血清白蛋白的通透量。结果:处理后30,60,120,240min,肿瘤坏死因子α干预组体外血脑屏障模型125Ⅰ-BSA通透量均高于其他组别(P<0.01),240min时达高峰;处理后30,60min,Y-27632预处理组125Ⅰ-BSA通透量低于肿瘤坏死因子α干预组(P<0.01),表现出拮抗肿瘤坏死因子α的作用,自120min后,Y-27632预处理组与Y-27632对照组比较,差异也有显著性意义(P<0.05)。结论:肿瘤坏死因子α可导致血脑屏障通透性增高,Y-27632预处理能早期逆转肿瘤坏死因子α对血脑屏障通透性的影响。     

丹参对类风湿关节炎患者滑膜细胞肿瘤坏死因子α表达的影响

目的探讨丹参对类风湿关节炎（RA）成纤维样滑膜细胞肿瘤坏死因子α（TNF-α）表达的影响。方法抽取RA患者关节液进行体外培养分离滑膜细胞,在细胞培养至3～5代时,给予丹参处理,在处理48h后收集培养上清,ELISA检测TNF-α的表达水平。结果当丹参浓度依次以0,6.25,12.5,25,50,100mg/L递增时,TNF-α浓度分别为（161.68±7.55）pg/ml、（161.68±8.79）pg/ml、（163.49±25.68）pg/ml、（137.33±35.98）pg/ml、（81.32±22.84）pg/ml和（86.89±28.48）pg/ml。丹参浓度为50mg/L和100mg/L时,与加入丹参前比较差异有统计学意义（P值分别为0.001和0.004）。结论丹参可以抑制RA滑膜细胞TNF-α的分泌,并且这一抑制作用可能是其用于RA治疗的理论基础。     

青藤碱对体外人外周血单个核细胞肿瘤坏死因子α表达的影响

目的观察青藤碱对体外培养人外周血单个核细胞TNF-α表达的影响。方法将分离的正常人外周血单个核细胞分成5组,分别给予0.9%氯化钠注射液、高剂量青藤碱、中剂量青藤碱、低剂量青藤碱和地塞米松;孵育48 h后,提取细胞培养液上清液,用放免法检测肿瘤坏死因子α的含量,用半定量RT-PCR法检测外周血单个核细胞中TNF-αmRNA的表达。结果高剂量青藤碱组和地塞米松组细胞培养液的上清液TNF-α含量明显低于0.9%氯化钠注射液组(P<0.05),高剂量青藤碱组的TNF-α的含量水平明显低于中、小剂量青藤碱组(P<0.05),且其外周血单个核细胞TNF-α的mRNA表达水平较0.9%氯化钠注射液组有显著下降(P<0.05)。结论抑制人外周血单个核细胞表达TNF-α可能是青藤碱治疗强直性脊柱炎的机制之一。     

肿瘤坏死因子-α拮抗剂治疗对强直性脊柱炎患者骨代谢指标的影响

目的：观察强直性脊柱炎（AS）患者骨代谢指标变化及接受TNF-α拮抗剂治疗对AS患者骨代谢指标的影响。方法测定25名AS高活动（ASDAS＞2.1）强直性脊柱炎患者炎症指标、血清骨钙素N端中分子（N-MID）、β胶原降解产物（？-Crosslaps）及总I型胶原氨基端前肽（P1NP）水平,计算ASDAS,后经TNF-α拮抗剂治疗12周后复查上述骨代谢指标。并进行分析。结果 AS治疗前组与对照组在年龄、性别方面差异无统计学意义（P＞0.05）。AS治疗前组血清？-Crosslaps、N-MID和P1NP水平明显高于健康对照组。经12周TNF-α治疗前后组间比较,AS治疗后组ASDAS评分、ESR和CRP指标均较AS治疗前组降低,TNF-α拮抗剂治疗后AS组在疾病活动度方面有明显改善;AS组治疗后？-Crosslaps显著降低（P＞0.05）, N-MID和P1NP变化无统计学意义（P＞0.05）。结论 TNF-α拮抗剂治疗不仅可改善AS疾病活动度,同时对AS患者骨吸收骨破坏进行有效的阻止。     

肺结核患者血清瘦素和肿瘤坏死因子-α检测及其相关性

目的检测肺结核患者血清瘦素(leptin)和肿瘤坏死因子-α(TNF-α)浓度并分析其相关性。方法采用放射免疫法测定48例活动性肺结核患者和30例正常人的血清瘦素和TNF-α。同时测定两组实验者的细胞免疫功能,分析瘦素与这些参数的相关性。结果肺结核患者的血清瘦素[(3.0±1.74)μg/L]水平显著高于正常对照组[(1.32±0.18)μg/L(P<0.01)]。而TNF-α血清浓度[(1.8±0.84)μg/L]也高于对照组[(0.79±0.26)μg/L](P<0.01),两组间无显著相关性。肺结核患者的T细胞亚群CD3+T、CD4+T、CD8+T和总淋巴细胞计数显著下降,且与血清瘦素呈负相关关系。结论肺结核患者的血清高瘦素水平可能参与了细胞免疫功能的降低。     

运动对胰岛素抵抗大鼠脂肪组织肿瘤坏死因子-α表达的影响

目的研究运动对高脂饮食诱导的胰岛素抵抗(IR)大鼠脂肪组织中肿瘤坏死因子-α（TNF-α）表达的影响。 方法将30只Wistar大鼠随机分为对照组和高脂组，分别给予基础饲料与高脂饲料喂养18周；将造模成功的高脂组IR大鼠随机分为静息组和运动组，均继续给予高脂饲料喂养，同时对运动组大鼠实施游泳训练，共持续6周。于实验进行24周时检测各组大鼠体重、血清空腹血糖（FBG）、空腹胰岛素(FINS)和游离脂肪酸(FFA)水平，并同时计算胰岛素敏感指数（ISI）；采用实时荧光定量聚合酶链反应、免疫印迹技术分别检测各组大鼠脂肪组织中TNF-α mRNA及蛋白表达水平。 结果喂养18周时发现高脂组ISI较对照组明显降低，提示高脂组IR模型制作成功；运动组大鼠经游泳训练6周后，其ISI较静息组明显升高（P<0.05），但仍显著低于对照组水平（P<0.01）；静息组大鼠脂肪组织中TNF-α mRNA及蛋白表达水平均较对照组明显升高（P<0.05）；运动组大鼠脂肪组织中TNF-α mRNA和蛋白表达较静息组进一步升高(P<0.05)。 结论高脂喂养可诱导大鼠产生IR，规律运动可减轻大鼠IR状态，其机制可能与上调脂肪组织中TNF-α表达有关。     

局灶性脑缺血预处理对大鼠肿瘤坏死因子表达影响的实验研究

目的观察局灶性缺血预处理对大鼠肿瘤坏死因子（TNF—a）的表达影响,探讨TNF—a与缺血耐受的关系及其在内源性保护机制中的作用。方法利用线栓法建立局灶性脑缺血耐受的动物模型。选用30只SD大鼠．随机将30只大鼠分为实验组：预缺血＋缺血（IP＋MCAO）;假手术组（SS＋MCAO）：假手术代替IP,余同实验组：对照组（SS＋SS）,每组10只。评价指标包括神经功能缺损评分、光镜下组织病理改变及免疫组织化学染色和图像分析比较各组TNF-a的表达变化。结果局灶性IP能够明显改善3d后的大鼠的神经功能评分,减轻组织学的损伤,下调了TNF—a的表达,IOD值实验组（8109．53±571．21）对比假手术组（10704．72±584．01）,差异有统计学意义（F=233．59,P〈0．05）。结论局灶性缺血预处理对随后的脑梗死有明显的保护作用．能够诱导缺血耐受的产生,其可能的机制是通过下调肿瘤坏死因子的表达。     

Effects of tumor necrosis factor-alpha on the in vitro maturation of tumor-reactive effector T cells

Culture methods that enhance the anti-tumor reactivity of primed T cells would be important in adoptive immunotherapy of cancer. Using several different syngeneic murine tumor models, the authors evaluated the effects of tumor necrosis factor-alpha (TNF-alpha) exposure on tumor-draining lymph node (TDLN) cells during in vitro activation. Mice were inoculated with weakly immunogenic (i.e., MCA 205, MCA 207 sarcoma) or the poorly immunogenic (i.e., D5 melanoma) tumor cells, and TDLN cells were harvested 9 or 10 days later for activation by an anti-CD3/interleukin-2 culture procedure. Human recombinant TNF-alpha (25 ng/mL) added during the activation culture resulted in a two-fold increase in interferon-gamma release (type 1 response) and a significant reduction of interleukin-10 (type 2 response) after tumor antigen stimulation. In an adoptive transfer model, TNF-alpha-cultured TDLN cells mediated significantly greater regression of established tumor than did TDLN cells cultured in the absence of TNF-alpha in five of five experiments. Neutralization of interleukin-10 monoclonal antibody further augmented the therapeutic efficacy of TNF-alpha-cultured TDLN cells. These studies document the ability of TNF-alpha to selectively promote a type 1 over a type 2 response in a bulk population of tumor-primed T cells during in vitro activation.     

Activation of the adenosine A3 receptor in RAW 264.7 cells inhibits lipopolysaccharide-stimulated tumor necrosis factor-alpha release by reducing calcium-dependent activation of nuclear factor-kappaB and extracellular signal-regulated kinase 1/2

Bacterial lipopolysaccharide (LPS) activates the immune system and promotes inflammation via Toll-like receptor (TLR) 4, which regulates the synthesis and release of tumor necrosis factor (TNF)-alpha and other inflammatory cytokines. Previous studies have shown that the nucleoside adenosine suppresses LPS-stimulated TNF-alpha release in human UB939 macrophages by activating an adenosine A(3) receptor (A(3)AR) subtype on these cells. In this study, we examined the mechanism(s) underlying A(3)AR-dependent inhibition of TNF-alpha release in a mouse (RAW 264.7) cell line. Treatment of RAW 264.7 cells with LPS (3 mug/ml) increased TNF-alpha release, which was reduced in a dose-dependent manner by adenosine analogs N(6)-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (IB-MECA) and R-phenylisopropyladenosine and reversed by selective A(3)AR blockade. The increase in TNF-alpha release was preceded by an increase in intracellular Ca(2+) levels. Inhibition of intracellular Ca(2+) release by IB-MECA, a selective agonist of the A(3)AR, or with BAPTA-AM, an intracellular Ca(2+) chelator, reduced LPS-stimulated TNF-alpha release. Activation of the A(3)AR or inhibition of intracellular Ca(2+) release also reduced LPS-stimulated nuclear factor-kappaB (NF-kappaB) activation and extracellular signal-regulated kinase 1/2 (ERK1/2) phosphorylation. Similar inhibition by A(3)AR was observed for LPS-stimulated inducible nitric-oxide synthase. These data support the contention that inhibition of LPS-stimulated release of inflammatory molecules, such as TNF-alpha and NO via the A(3)AR, involves suppression of intracellular Ca(2+)signaling, leading to suppression of NF-kappaB and ERK1/2 pathways.     

Effects of ultrasound on the structure and function of tumor necrosis factor-alpha

The objective of this study was to investigate the effects of ultrasound on the structure and function of human tumor necrosis factor-alpha (TNF-alpha) and to study whether TNF-alpha underwent a denaturation process and the molecular structure was damaged when it was irradiated by ultrasound. The samples of TNF-alpha were dissolved in aqueous solution and filled into polystyrene tubes. High intensity ultrasound processor (20 kHz frequency, burst mode, 0.5 duty factor, 100-500 W total electrical power, 0-20 min total treatment time) was used during the treatment. The biologic activity of TNF-alpha was determined by its toxic activity towards TNF-alpha sensitive cell line L929 in the presence of actinomycin D. The methods of sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF-MS) were used to detect the integrity of TNF-alpha molecule after it was irradiated by ultrasound. The results showed TNF-alpha could keep its biological activity, instead of undergoing a denaturation process, when it is irradiated by ultrasound in the aqueous solution; at the same time, the aggregates of TNF-alpha formed by the recombinant DNA E. coli could be dissociated through the molecular vibration induced by ultrasound energy. The biologic activity of TNF-alpha was not reduced, but small quantities of TNF-alpha molecular structure were damaged during the process of sonication. These features of TNF-alpha molecule irradiated by ultrasound probably gave TNF-alpha the advantage in being used in the drug microencapsulation and provided a new drugs formulation for tumor therapy.     

小鼠胚胎成纤维细胞成熟化过程中肿瘤坏死因子α的作用(英文)

背景:近年的研究表明,肿瘤坏死因子α对不同组织成纤维细胞的作用具有组织特异性及浓度依赖性.目的:观察肿瘤坏死因子α及其信号传导途径中特异性激酶抑制剂对小鼠胚胎成纤维细胞成熟化所起的作用.方法:体外培养小鼠胚胎成纤维细胞,将细胞分为3组:第1组用含体积分数2%血清的DMEM高糖培养基培养作为空白对照组;第2组用含100 μg/L肿瘤坏死因子α的培养基培养;第3组先加入质量浓度为50 μg/L的Anti-TNFRSF1B作用1 h后,倒出培养基再加入含有肿瘤坏死因子α的培养基继续培养.采用RT-PCR法测定各组Ⅰ型胶原蛋白和基质金属蛋白酶3 mRNA表达、Western Blot法测定各组Ⅰ型胶原蛋白和基质金属蛋白酶3蛋白表达.结果与结论:小鼠胚胎成纤维细胞在一定质量浓度肿瘤坏死因子α作用下,其信号传导途径特异性激酶发生磷酸化或蛋白被激活,信号通路被激活,促进基质金属蛋白酶3的活化,明显降低Ⅰ型胶原的表达.加入其信号传导途径的抑制剂Anti-TNFRSF1B后,肿瘤坏死因子α的效应得到了一定的抑制,但并未完全消除,这更进一步证明肿瘤坏死因子α对小鼠胚胎成纤维细胞活化的作用.     

小分子干扰RNA真核表达载体阻断U937细胞肿瘤坏死因子α基因的表达

背景:无菌性松动是导致人工关节置换后失败和降低人工关节使用寿命的主要原因,肿瘤坏死因子α是一个极具吸引力的无菌性松动治疗新靶点.小分子干扰RNA作为一种新型的基因阻断技术,被广泛用于新基因筛选、基因功能鉴定、信号转导研究以及基因治疗方面,显示出广阔的应用前景.目的:构建人肿瘤坏死因子α基因的小分子干扰RNA真核表达载体,观察其对U937细胞中肿瘤坏死因子α基因表达的干涉作用.方法:将合成的2对小分子干扰RNA寡核苷酸链分别退火形成双链,连接入pSilencer4.1-CMV neo真核表达载体,分别命名为pSilencer-T1和pSilencer-T2,经酶切及测序鉴定.电转染法转染重组质粒入U937细胞,G418筛选后反转录-聚合酶链反应检测其对肿瘤坏死因子α基因mRNA的干涉效果.结果与结论:经酶切及测序鉴定,成功构建了siRNA真核表达载体,经电转染法转染U937细胞后,反转录-聚合酶链反应显示所构建的干涉肿瘤坏死因子α基因的真核表达载体成功地抑制了目的基因的表达,U937细胞中肿瘤坏死因子α基因的mRNA表达水平明显降低.提示成功构建了人肿瘤坏死因子α基因的RNA干涉真核表达载体pSilencer-T1和pSilencer-T2,并在U937细胞中有效地发挥了对肿瘤坏死因子α基因表达的干涉作用.     

核因子-κB对急性胰腺炎时肿瘤坏死因子-αmRNA表达的调控

目的观察急性胰腺炎(acutepancreatitis,AP)发生时胰腺组织中核因子κB(NFκB)对肿瘤坏死因子αmRNA表达的调控作用。方法Wistar大鼠64只,随机均分为AP组和对照组,AP组以蛙皮素(Caerulein)腹腔内注射制成AP模型。分别于12h、24h、36h、72h后处死实验动物,用流式细胞术(FCM)检测NFκB激活水平,用半定量RTPCR检测TNFαmRNA的表达水平,并分析二者之间的相关性。结果AP组的NFκB激活及TNFαmRNA表达水平在各时间点均显著高于同时段对照组的水平(P<0.01),二者之间具有相关性(P<0.05)。结论AP发生时,胰腺组织中NFκB高度活化,促进了TNFαmRNA的表达,从而加重了胰腺的损伤。     

Bacterial translocation and tumor necrosis factor-alpha gene expression in experimental hemorrhagic shock

OBJECTIVE: To investigate whether bacterial translocation is the causative mechanism underlying cytokine production during hemorrhagic shock. DESIGN: Prospective, randomized, unblinded animal study. SETTING: Surgical research laboratories of Shiga University of Medical Science. SUBJECTS: Male Sprague-Dawley rats. INTERVENTIONS: The rats were randomly divided into three groups. Each animal was anesthetized with pentobarbital, given a continuous infusion of 0.9% saline, and monitored for blood pressure. The normoxic and sham shock groups breathed room air, whereas the hyperoxic shock group was administered 100% oxygen. Except in the sham shock group, blood was withdrawn to induce a hemorrhagic shock state, then the shed blood was reinfused. Sixty minutes after the induction of hemorrhagic shock, arterial blood cultures were performed in all three groups. The animals were then killed, and their mesenteric lymph nodes (MLNs) were harvested for bacterial culture. The terminal ileum, liver, spleen, kidney, lung, and MLNs were also collected for histologic study by in situ hybridization. MEASUREMENTS AND MAIN RESULTS: In the bacteriologic study, the prevalence of bacterial translocation was 0% (0/11) in the hyperoxic shock group, 55% (6/11) in the normoxic shock group, and 0% (0/9) in the sham shock group. In the in situ hybridization study, tumor necrosis factor-alpha gene expression was detected only in the ileal tissue, MLNs, and spleens of the normoxic shock group. Blood cultures were sterile in all three groups. CONCLUSIONS: Bacterial translocation occurred in MLNs within 1 hr of hemorrhage. Hemorrhagic shock causes tumor necrosis factor-alpha gene expression as well as bacterial translocation in MLNs, but not in the liver, in this model. Bacterial translocation was prevented by hyperoxia early in the course of hemorrhagic shock. Hyperoxia also prevented tumor necrosis factor-alpha gene expression along the bacterial invasion route.