人H7N9禽流感病毒、高致病H5N1禽流感病毒及H1N1流感病毒感染小鼠特征分析

目的对比分析人高致病H5N1禽流感病毒、H7N9禽流感病毒及H1N1流感病毒分别感染BALB/c小鼠后的机体反应特征。方法分别以H7N9病毒、H5N1病毒和H1N1病毒滴鼻感染BALB/c小鼠,观察小鼠存活率、体征变化及感染后肺组织病理损伤差异,检测小鼠感染流感病毒后肺组织增殖细胞核抗原(PCNA)表达并观察小鼠感染后修复状况。结果 H7N9病毒、H5N1病毒和H1N1病毒均感染BALB/c小鼠,小鼠存活率依次为H7N9H1N1H5N1,肺组织病理损伤严重程度依次为H5N1H1N1H7N9,PCNA表达水平依次为H7N9H1N1H5N1。结论 H7N9病毒感染后宿主炎症反应较小,感染后小鼠肺组织自我修复能力较强;H5N1病毒感染BALB/c小鼠后的机体反应最为强烈,感染后恢复能力差,致死率高。     

人H7N9禽流感病毒与H1N1流感病毒感染小鼠病理学损伤的比较

目的比较分析H7N9病毒与H1N1病毒感染小鼠病理学损伤特点,初步探讨两种病毒感染致小鼠急性肺损伤的致病机制。方法 H7N9病毒与H1N1病毒分别感染小鼠,观察不同病毒感染后小鼠生存率,并于不同时间点取心、肝、脾、肺、肾、脑、肠等组织,伊红-苏木素染色并进行组织病理学分析,免疫组化检测病毒抗原分布及中性粒细胞浸润。综合分析肺组织病理损伤与病毒复制、宿主免疫反应之间的关系。结果 H7N9病毒感染小鼠肺及脾脏损伤较轻,存活率较高。H1N1病毒感染的小鼠肺及脾脏损伤较重,感染后9 d全部死亡;两种病毒抗原主要分布于支气管上皮细胞、少量间质细胞和肺泡上皮细胞,病毒复制水平无明显差异。但H1N1病毒感染后肺及脾脏中均有大量中性粒细胞浸润,小鼠机体炎症反应明显强于H7N9病毒感染后小鼠炎症反应。结论 H7N9病毒与H1N1病毒感染后小鼠病理学损伤特点及程度均不同,病毒复制是小鼠肺损伤的诱发因素但并非决定因素,宿主针对病毒感染产生的免疫反应程度与急性肺损伤密切相关。     

遗传位点影响小鼠对约氏疟原虫易感性的研究

探讨Listr1遗传位点影响小鼠对疟原虫易感性的可能性及其初步作用机制。致死型约氏疟原虫(Plasmodium yoelii 17XL,P.y17XL)感染C.B6By Listr1小鼠（BALB/c小鼠基因背景下引入C57BL/6小鼠Listr1遗传位点的同类系小鼠）和BALB/c小鼠,分析二者生存率和感染率的差异;HE染色分析P.y17XL感染后第5天C.B6By Listr1小鼠和BALB/c小鼠肝脏组织损伤情况;定量PCR法检测P.y17XL感染后小鼠肝脏组织第1、2、5天IL-1β、IL-6、TNF-α、IFN-γ的表达含量。结果显示,P.y17XL感染后4~8 d C.B6By Listr1小鼠虫血症水平显著高于BALB/c小鼠（P＜0.05）。C.B6By Listr1小鼠于感染后7~9 d全部死亡;而半数BALB/c小鼠于感染后8~9 d死亡,其余BALB/c小鼠至感染后第13天仍存活,两种小鼠生存率差异具有统计学意义（P＜0.01）。C.B6By Listr小鼠P.y17XL感染后第5天肝组织HE染色可见到明显的疟色素沉积;而BALB/c小鼠全片基本未见到疟色素的沉积。P.y17XL感染后第2天BALB/c小鼠肝组织IL-6（P＜0.05)、TNF-α（P＜0.05)mRNA水平显著高于C.B6By Listr1小鼠。结果表明Listr1遗传位点可以影响小鼠对疟原虫的易感性,与肝脏固有免疫相关。     

Flk1+间充质干细胞减轻四氯化碳导致的肝纤维化的研究

许多慢性肝脏疾病都会发生肝纤维化,但是目前尚缺乏对肝纤维化切实有效的治疗手段。实验发现,Flk1(fetalliverkinase)阳性间充质干细胞(MSC)能够减轻四氯化碳(CCl₄ )所致小鼠肝纤维化。取雄性BALB c小鼠骨髓,分离培养Flk1⁺ MSC ,用CCl₄ 制作雌性小鼠肝纤维化模型,在CCl₄ 损伤后立即或1周后经尾静脉注射Flk1⁺ MSC ,2或5周后检测受体小鼠肝脏的纤维化程度和供体细胞的植入。结果发现,CCl₄ 损伤后立即注射Flk1⁺ MSC ,可以使肝脏损伤程度明显减轻,减少胶原沉积,使肝脏羟脯氨酸含量及血清纤维化指标显著下降;而损伤1周后注射细胞则无明显变化。免疫荧光、PCR和荧光原位杂交方法证实,在受体肝脏中有供体细胞植入,呈上皮细胞形态,并表达白蛋白,但是数量很少。因此,Flk1⁺ MSC具有潜在的植入肝组织的能力,并可能启动肝组织的内源性修复,减轻CCl₄ 导致的肝纤维化。     

过氧化氢对培养心肌细胞损伤作用的研究

氧化应激时产生大量的自由基,造成心肌细胞的损伤.过氧化氢(H₂O₂)是有机体氧化代谢产物,同时是一种活性氧.应用不同浓度的H₂O₂,分别于不同作用时间,动态观察其对心肌细胞的损伤作用.从实验结果看到,低浓度的H₂O₂（＜0.1 mmol/L）作用2 h,使心肌细胞产生早期的生物化学的改变,如MDA产生堆积和细胞周期时相改变（G1期细胞增加,G2期细胞减少）,此时心肌酶基本无泄漏,心肌细胞的死亡率很低,HE形态学观察基本无改变;随着H₂O₂浓度的增加（1～5 mmol/L）和作用时间的延长,进一步诱导细胞损伤加剧,LDH释放和MDA积累明显升高,细胞死亡率也明显增加,已具有统计学意义.同时可观察到其病理形态学的坏死性改变;当10 mmol/L H₂O₂作用时,细胞大量死亡,形态学可见细胞极度收缩、脱落,形成大面积的细胞脱失区.因此,H₂O₂作为一种活性氧自由基,依其浓度和作用时间不同可造成不同程度的心肌细胞的损伤.辣根过氧化物酶作为一种自由基清除剂,可明显减少H₂O₂活性氧自由基对心肌细胞的损伤作用.     

NO和氧自由基在心肌缺血再灌注损伤中的协同作用——心肌缺血再灌注损伤产生的NO自由基的ESR研究

用低温电子自旋共振(ESR)技术检测到了大鼠心肌缺血再灌注过程产生的NO自由基与含铁蛋白结合的ESR信号 并且利用这一技术研究了大鼠心肌缺血再灌注损伤过程中NO和氧自由基的协同作用.结果发现,在缺血再灌注损伤的心肌中可同时检测到氧自由基和与血红蛋白β-亚基铁结合的NO自由基(β-NO复合物).在正常心肌中检测不到这两个信号,即使在灌注液中加入L-精氨酸也检测不到这两个信号.在缺血再灌注损伤的心肌中就可以检测的这两个信号了,而且随着在灌注液中加入L-精氨酸浓度的增加,这一信号也随之增加.在灌注液中加入NO合成酶抑制剂N~G-硝基精氨酸甲脂(NAME),这两个信号受到抑制.在灌注液中检测标志心肌损伤的乳酸脱氢酶(LDH)和肌酸激酶(CK)活性发现,在灌注液中加入低浓度的L-精氨酸(1mmol/L以下),对缺血再灌注心肌损伤有一定保护作用,但是,若加入高浓度L-精氨酸,则加重缺血再灌注心肌的损伤.加NAME对缺血再灌注心肌有明显保护作用.在灌注液中加入黄嘌吟/黄嘌吟氧化酶(X/XO)或Fe²⁺/H₂O₂,同时增加缺血再灌注心肌中的NO和氧自由基含量,并加重心肌的损伤.在灌注液中加入超氧化物歧化酶(SOD)和过氧化氢酶(CAT),同时减少缺血再灌注心肌中NO和氧自由基的含量,并减轻心肌的损伤.     

促甲状腺激素单克隆抗体的制备

获得了抗促甲状腺激素(TSH)单克隆抗体杂交瘤细胞20株,其中T₇₄A₁₀小鼠腹水滴度为1:50 000,亲和常数为7.15×10⁹L/mol,T₇₁B₁₁小鼠腹水滴度为1:150000,亲和常数为8.75×10⁹L/mol.两个抗体与人绒毛膜促性腺激素(HCG)、促卵泡激素(FSH)和促黄体生成激素(LH)的交叉反应分别小于1.1×10^-6%、0.01%和0.016%.将T₇₄A₁₀和T₇₁B₁₁应用于TSH免疫放射分析中,得到了满意的结果.     

甲型H1N1流感病毒感染小鼠的发病机制

目的甲型H1N1流感病毒A/California/7/2009感染BALB/c小鼠,研究甲型H1N1流感病毒病毒性肺炎发病机制。方法 4～6周龄雌性BALB/c小鼠60只,随机分为2组,实验组和对照组,每组30只。CA7流感病毒滴鼻制备甲流病毒感染小鼠模型。攻毒后第5天解剖实验和对照组小鼠,取肺组织,测定肺组织中IL-2,IL-6,TNF-α含量。结果结果实验组肺组织中IL-6,TNF-α,水平明显高于对照组,IL-2水平明显低于对照组,差异均有显著性（P〈0.05）。结论 IL-6、TNF-α、IL-2这3种细胞因子在感染甲流病毒后的显著性变化与病毒感染后的肺组织病理损伤有密切的关系。     

抗人IL-6单克隆抗体的制备及鉴定

用基因重组人IL-6免疫Balb/c小鼠,采用小鼠杂交瘤技术,筛选克隆到分泌抗人重组IL-6单克隆抗体的杂交瘤细胞株,并对其中2H₂、 1D₂ 和4B₄瘤细胞株进行了鉴定.其抗体类别均为IgG,亚类分别为IgG1和IgG2a.用多种细胞因子和无关蛋白的鉴别试验结果证实它们均特异地识别rhIL-6.免疫转染结果显示,该单抗识别分子质量为21 ku的IL-6单一条带.IL-6单克隆抗体的亲和常数K_aff= 1.62×10⁹ (mol/L)^-1.     

线粒体参与氧自由基代谢的功能

根据呼吸链中传递的电子可从细胞色素c漏出传给H2O2的实验结果,结合文献关于呼吸链底物端电子漏导致生成O2·-和H2O2的报导,提出了呼吸链主链电子传递之外,存在一个由两类电子漏衔接形成的电子漏路径.由于这一路径实质上是氧自由基的代谢途径而显示出线粒体可能具有参与氧自由基代谢的功能.     

In vivo evidence of free radical generation in the mouse lung after exposure to Pseudomonas aeruginosa bacterium: An ESR spin-trapping investigation

Abstract

In the Pseudomonas aeruginosa-induced rodent pneumonia model, it is thought that free radicals are significantly associated with the disease pathogenesis. However, until now there has been no direct evidence of free radical generation in vivo. Here we used electron spin resonance (ESR) and in vivo spin trapping with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone to investigate free radical production in a murine model. We detected and identified generation of lipid-derived free radicals in vivo (a^N =14.86±0.03 G and a^H_β =2.48±0.09 G). To further investigate the mechanism of lipid radical production, we used modulating agents and knockout mice. We found that with GdCl₃ (phagocytic toxicant), NADPH-oxidase knockout mice (Nox2^?/^?), allopurinol (xanthine-oxidase inhibitor) and Desferal (metal chelator), generation of lipid radicals was decreased; histopathological and biological markers of acute lung injury were noticeably improved. Our study demonstrates that lipid-derived free radical formation is mediated by NADPH-oxidase and xanthine-oxidase activation and that metal-catalysed hydroxyl radical-like species play important roles in lung injury caused by Pseudomonas aeruginosa.     

The effects of oxygen radical — mediated pulmonary endothelial damage on cancer metastasis

Summary The vascular bed of the lung is susceptible to environmental and host-mediated injury from free radicals. The lung is also a frequent site for the formation of cancer metastases. Since the circulation is important for the spread of cancer and because the endothelium is a barrier between the circulation and extravascular tissue, we have postulated that free radical damage to the pulmonary microvasculature enhances the formation of metastases. Pulmonary endothelial injury was induced in mice by bleomycin (120 mg/kg i.v.) or by exposure to 90% oxygen for 2–4 days. In rats, damage was elicited by intravenous injection of cobra venom factor which activates the circulating leukocytes. Endothelial damage was demonstrated by morphology and by measurement, in lung lavage fluids, of increased protein and/or leakage of ¹²⁵I-albumin, previously injected intravenously. When radiolabeled cancer cells were injected into the tail vein during periods of pulmonary endothelial damage, there was a 3–36 fold increase in the numbers of these cells located in the lung after 24 hours. Subsequently more metastatic tumors formed in the animals with injured lungs. In rats, the enhanced localization was prevented by pretreatment of the animals with catalase or with antineutrophil antibodies. We have also demonstrated that stimulation of rat cancer cells by the chemotactic peptide N-fMLP is followed by chemiluminescence, amplified in the presence of luminol. Evidence for the generation of oxygen radicals by these cells includes inhibition of the response in the absence of oxygen or in the presence of superoxide dismutase, catalase, and mannitol, and dose-dependent reduction of acetylated cytochrome C. We conclude that free radical-mediated damage to the pulmonary endothelium significantly increases the metastasis of circulating tumor cells and we postulate that some cancer cells may directly facilitate their spread by generating free radicals.     

Synergic effects of NO and oxygen free radicals in the injury of ischemia-reperfused myocardium——ESR studies on NO free radicals generated from ischemia-reperfused myocardium

The ESR signal of NO bound to hemoglobin was detected during the ischemia-reperfusion of myocardium with low temperature ESR technique, and the synergic effects of NO and oxygen free radicals in the injury of the process were studied with this technique. Oxygen free radicals and NO bound to β-subunit of hemoglobin (β-NO complex) could be detected simultaneously in the ischemia-reperfused myocardium. Those signals could not be detected from the normal myocardium even in the presence of L-arginme. However, those signals could be detected and were dose-dependent with L-arginine in the ischemia-reperfused myocardiums and the signal could be suppressed with the inhibitor of NO synthetase, NG-nitro-L-arginine methylester (NAME). Measurement of the activities of lactate dehydrogenase (LDH) and creatine kinase (CK) in the coronary artery effluent of ischemia-reperfused heart showed that L-arginine at lower concentration (<1 mmol/L) could protect the heart from the ischemia-reperfusion injury but at higher con     

Chemical reactivity of the carbon-centered free radicals and ferrous iron in coals: Role of bioavailable Fe2+ in coal workers' pneumoconiosis

Striking differences in the prevalence of coal workers' pneumoconiosis (CWP) exist between different coal mine regions. The major factors responsible for the observed regional differences in CWP have not yet been identified. In the present study, chemical reactivity of the carbon-centered free radicals in coals and lung tissues, as well as ferrous iron in the coals, were studied by ESR techniques. The ESR spectra clearly demonstrated the presence of at least two types of carbon-centered free radical species, which might respectively attribute to the macromolecular phase and the molecular phase of coal. Grinding produced free radicals in coals. Exposure of freshly ground coal to air for 28 h induced a slight increase of free radicals for most of the coals, and a slight decrease after 4 months' exposure. The lung tissue samples of coal workers deceased of CWP showed similar ESR spectra as coal samples, and these radicals were highly stable in the lung. After incubation of coals with glutathione, hydrogen peroxide, sodium formate or oxygen, the coal sample from the Gardanne mine which has never induced CWP, and thus is the least hazardous coal, showed the most significant change in the carbon-centered free radical concentration. No significant changes were observed among other coals reported to induce CWP. On the other hand, we found that the coals released different amounts of Fe²⁺ in an acidic medium. Interestingly, the prevalence of CWP correlates positively with the released Fe²⁺ content in these coals and with the amount of oxygen radicals produced by the interaction of Fe²⁺ with O₂ in the acidified coal filtrates. Our studies indicate that the carbon-centered free radicals may not be biologically relevant to coal dust-induced pneumoconiosis, whereas the acid soluble Fe²⁺, which may be dissolved in the phagolysosomes of macrophages, can then lead to Fe²⁺-induced oxidative stress and eventual CWP development.     

In vivo evidence of free radical generation in the mouse lung after exposure to Pseudomonas aeruginosa bacterium: an ESR spin-trapping investigation

In the Pseudomonas aeruginosa-induced rodent pneumonia model, it is thought that free radicals are significantly associated with the disease pathogenesis. However, until now there has been no direct evidence of free radical generation in vivo. Here we used electron spin resonance (ESR) and in vivo spin trapping with α-(4-pyridyl-1-oxide)-N-tert-butylnitrone to investigate free radical production in a murine model. We detected and identified generation of lipid-derived free radicals in vivo (a(N) =14.86 ± 0.03 G and a(H)(β) =2.48 ± 0.09 G). To further investigate the mechanism of lipid radical production, we used modulating agents and knockout mice. We found that with GdCl(3) (phagocytic toxicant), NADPH-oxidase knockout mice (Nox2(-)/(-)), allopurinol (xanthine-oxidase inhibitor) and Desferal (metal chelator), generation of lipid radicals was decreased; histopathological and biological markers of acute lung injury were noticeably improved. Our study demonstrates that lipid-derived free radical formation is mediated by NADPH-oxidase and xanthine-oxidase activation and that metal-catalysed hydroxyl radical-like species play important roles in lung injury caused by Pseudomonas aeruginosa.     

Bulky DNA-adduct formation induced by Ni(II) in vitro and in vivo as assayed by 32P-postlabeling

Various small oxidation products (e.g. 8-hydroxydeoxyguanosine) can be induced in DNA by nickel compounds. In this study, the ³²P-postlabeling assay was applied to determine whether Ni(II) compounds are able to induce bulky DNA-adduct formation in vitro and in vivo. In vitro studies detected two major and several minor adducts in DNA incubated with NiCl₂ and H₂O₂ at 37°C for 1 h. Formation of the two major adducts increased with incubation time (0–24 h) and NiCl₂ concentration (0–800 μM). Adduct levels were greatly reduced by hydroxyl free-radical scavengers, i.e. 0.4 M sodium formate or 0.05 M p-nitrosodimethylaniline, and by a singlet oxygen scavenger, 0.05 M sodium azide. The in vitro effects of NiCl₂ on DNA were significantly enhanced by (1) addition of 3 mM ascorbic acid, (2) replacement of H₂O with D₂O in the reaction, and (3) prior denaturation of DNA. Adduct formation presumably involved a Fenton-type reaction, in which DNA crosslinks may arise by reaction with hydroxyl free radicals and singlet oxygen.For in vivo studies, male 6–8 wk old B6C3F1 mice were used. In untreated mice, several I-compounds (putative indigenous DNA modifications that increase with age) were detected in liver, kidney, and lung. Two of these spots (1 and 2) were chromatographically identical to the two major spots induced by Ni(II) in vitro. The intensities of spots 1 and 2 in kidney and of some other spots in liver and lung were increased 1 and 2 h after i.p. injection with a single dose of 170 μmoles/kg NiAc₂. The effects of NiAc₂ were reduced or undetectable in the three tissues 24 h after treatment. These observations indicate the capacity of Ni(II) to induce and modulate bulky DNA modifications both in vitro and in vivo.     

Vascular effects of oxygen-derived free radicals

This review attempts to summarize the available data regarding the vascular actions of free oxygen radicals. Studies on blood vessels in situ and in vitro demonstrate that free oxygen radicals can evoke both vasodilation and vasoconstriction. Free oxygen radicals can modulate the tone of vascular smooth muscle by acting directly on the smooth muscle cells, and also via indirect mechanisms by changes in the production or biological activity of vasoactive mediators. The individual oxygen radicals may have different (sometimes opposite) vascular effects. Superoxide anion inactivates endothelium-derived relaxing factor and the adrenergic neurotransmitter norepinephrine. Hydrogen peroxide and the hydroxyl radical evoke vasodilation by acting directly on vascular smooth muscle and also by stimulating the synthesis/release of endothelium-derived relaxing factor. In acute arterial hypertension or experimental brain injury oxygen radicals are important mediators of vascular damage. Production of oxygen-derived free radicals by activated neutrophils may be responsible for vasodilation and increased permeability of capillary membrane during the acute inflammatory process. Free oxygen radicals also play an important role in reperfusion injury of various organs, and vascular actions of the free radicals may contribute to the damage of parenchymal tissues.     

Disruption of cytochrome P4501A2 in mice leads to increased susceptibility to hyperoxic lung injury

Hyperoxia contributes to acute lung injury in diseases such as acute respiratory distress syndrome. Cytochrome P450 (CYP) 1A enzymes have been implicated in hyperoxic lung injury, but the mechanistic role of CYP1A2 in pulmonary injury is not known. We hypothesized that mice lacking the gene Cyp1a2 (which is predominantly expressed in the liver) will be more sensitive to lung injury and inflammation mediated by hyperoxia and that CYP1A2 will play a protective role by attenuating lipid peroxidation and oxidative stress in the lung. Eight- to ten-week-old WT (C57BL/6) or Cyp1a2^−/− mice were exposed to hyperoxia (>95% O₂) or maintained in room air for 24–72 h. Lung injury was assessed by determining the ratio of lung weight/body weight (LW/BW) and by histology. Extent of inflammation was determined by measuring the number of neutrophils in the lung as well as cytokine expression. The Cyp1a2^−/− mice under hyperoxic conditions showed increased LW/BW ratios, lung injury, neutrophil infiltration, and IL-6 and TNF-α levels and augmented lipid peroxidation, as evidenced by increased formation of malondialdehyde– and 4-hydroxynonenal–protein adducts and pulmonary isofurans compared to WT mice. In vitro experiments showed that the F₂-isoprostane PGF₂-α is metabolized by CYP1A2 to a dinor metabolite, providing evidence for a catalytic role for CYP1A2 in the metabolism of F₂-isoprostanes. In summary, our results support the hypothesis that hepatic CYP1A2 plays a critical role in the attenuation of hyperoxic lung injury by decreasing lipid peroxidation and oxidative stress in vivo.     

Aerosolized bovine lactoferrin reduces lung injury and fibrosis in mice exposed to hyperoxia

This study investigated the ability of aerosolized bovine lactoferrin (bLF) to protect the lungs from injury induced by chronic hyperoxia. Female CD-1 mice were exposed to hyperoxia (FiO₂ = 80 %) for 7 days to induce lung injury and fibrosis. The therapeutic effects of bLF, administered via an aerosol delivery system, on the chronic lung injury induced by this period of hyperoxia were measured by bronchoalveolar lavage, lung histology, cell apoptosis, and inflammatory cytokines in the lung tissues. After exposure to hyperoxia for 7 days, the survival of the mice was significantly decreased to 20 %. The protective effects of bLF against hyperoxia were further confirmed by significant reductions in lung edema, total cell numbers in bronchoalveolar lavage fluid, inflammatory cytokines (IL-1β and IL-6), pulmonary fibrosis, and apoptotic DNA fragmentation. The aerosolized bLF protected the mice from oxygen toxicity and increased the survival fraction to 66.7 % in the hyperoxic model. The results support the use of an aerosol therapy with bLF in intensive care units to reduce oxidative injury in patients with severe hypoxemic respiratory failure or chronic obstructive pulmonary disease.