香烟烟雾吸入致大鼠肺与气管上皮细胞RAGE和S100A6表达增加

目的观察香烟烟雾长期染毒大鼠肺组织、气管上皮细胞中晚期糖基化终产物受体（RAGE）和钙结合蛋白（S100A6）的表达。方法健康雄性Wistar大鼠18只,随机分为对照组、低（烟雾浓度20%）和高（烟雾浓度60%）浓度组,每组6只。动式气体染毒装置每天染毒2次,每次30min,一周5d,共染毒43周。分离大鼠气管和肺组织,分离原代上皮细胞,收集并提取总蛋白;Westernblot方法检测RAGE和S100A6蛋白的表达。同时测定正常的人支气管上皮细胞株BEAS-2B和肺癌细胞株A549中2种蛋白的表达情况。结果气管组织贴块培养法分离出的上皮细胞纯度为（93.56±1.48）%;烟雾染毒组大鼠肺组织及气管上皮细胞中RAGE和S100A6蛋白的表达均显著增高。同时,肺癌细胞A549中RAGE及S100A6蛋白的表达量明显高于正常人支气管上皮细胞BEAS-2B。结论RAGE和S100A6有望作为香烟烟雾暴露致肺损伤的生物学标志。     

香烟烟雾暴露大鼠肺组织中RAGE蛋白表达的改变

[目的]观察香烟烟雾暴露大鼠肺组织中晚期糖基化终产物受体(receptor for advanced glycation endproducts,RAGE)蛋白的表达。[方法]健康雄性Wistar大鼠20只,随机分为3个香烟烟雾染毒组和1个对照组,每组5只。采用动式气体染毒装置对大鼠进行香烟烟雾的染毒,装置内烟雾及氧气浓度分别控制在20%和21%,每天染毒2次,每次30min,3个染毒组分别暴露1、2、4个月作为短、中、长时间暴露组。染毒结束后取动物肺脏,左肺甲醛固定作病理切片,右肺提取蛋白,分别采用免疫组织化学方法和免疫印迹方法(Western blot)定性和定量检测RAGE蛋白在各组大鼠肺组织中的表达。同时检测肺组织氧化损伤指标--线粒体细胞色素C氧化酶(cytochrome C oxidase,COX)的活性,并分析RAGE的表达与COX活性的相关性。[结果]免疫组织化学结果显示RAGE蛋白棕黄色阳性颗粒在香烟烟雾暴露组大鼠的肺泡上皮细胞、支气管上皮细胞以及淋巴细胞中均呈现阳性表达。Western blot结果显示,与对照组比较,中、长时间暴露组大鼠肺组织中RAGE蛋白的表达明显增高(P<0.01)。...     

miR-3666在香烟烟雾诱导人支气管上皮细胞恶性转化中的表达变化及其功能研究

目的 探索microRNA-3666(miR-3666)在香烟烟气长期染毒致使的人永生化支气管上皮细胞BEAS-2B恶性转化过程中的表达调控及其功能研究。方法 将BEAS-2B细胞以每孔1×10⁵个的浓度种植于六孔板Transwell膜上室，设置细胞气体染毒装置染毒程序为烟雾浓度20%,染毒时间10 min,每4天染毒一次，2次染毒后收集细胞作为染毒1代，依次获得染烟恶性转化第30代(S30)细胞。通过荧光定量PCR(qPCR)技术检测miR-3666在S30恶性转化细胞以及肺腺癌(LUAD)细胞系A549和H1299中的表达量；通过miRNA mimic转染，过表达S30及LUAD细胞系中的miR-3666;通过CCK8法检测过表达miR-3666后S30细胞和LUAD细胞系恶性增殖能力的变化；通过Transwell侵袭实验检测过表达miR-3666对S30细胞及LUAD细胞系侵袭能力的影响；通过流式细胞术检测miR-3666表达对S30细胞及LUAD细胞系凋亡能力的影响。结果 与BEAS-2B细胞相比，染烟恶性转化S30细胞和肺腺癌细胞系中miR-3666表达...     

香烟烟雾提取物对A549细胞β防御素-2表达的影响

目的研究香烟烟雾提取物（cigarette smoke extract,CSE）对人肺腺上皮细胞A549卢防御素-2（human β-defensin2,hBD-2）表达的影响。方法培养A549细胞,加或不加LPS（10艘刖）情况下用5％CSE进行干预。用RT—PCR法和Westem Blot法分别检测hBD-2mKNA和蛋白表达变化。结果LPS可以诱导A549细胞hBD-2的表达;5％CSE不影响A549细胞hBD-2的表达,但可抑制LPS的诱导作用。结论CSE可以抑制LPS诱导的肺腺上皮细胞hBD-2表达,这可能与CSE减弱呼吸道抵御外界微生物入侵能力。导致呼吸道感染及慢性阻塞性肺疾病有关。     

香烟烟雾暴露对大鼠睾丸转铁蛋白mRNA及蛋白表达水平的影响

目的探讨香烟烟雾暴露对大鼠睾丸结构及睾丸组织转铁蛋白(Tf)mRNA及其蛋白表达水平的影响。方法清洁级成年雄性SD大鼠160只,随机分为对照组及低(10根香烟)、中(20根香烟)、高(30根香烟)剂量染毒组,各染毒组大鼠分别染毒2、4、6、8、12周,共16组,每组10只。染毒组大鼠采用呼吸道静式染毒,1次/d,30 min/次。观察大鼠睾丸组织结构,采用RT-PCR及Western blot法检测睾丸组织Tf mRNA及其蛋白表达水平。结果染毒组大鼠睾丸病理切片可见生精上皮层次减少,生精细胞排列疏松,支持细胞空泡化。染毒4、8、12周时高剂量组大鼠睾丸组织Tf mRNA表达水平低于对照组和低剂量组,染毒8周时高剂量组大鼠睾丸组织Tf蛋白表达水平低于对照组,染毒12周时中、高剂量组大鼠睾丸组织Tf蛋白表达水平低于低剂量组,差异均有统计学意义(P0.05)。结论香烟烟雾暴露可导致雄性大鼠睾丸组织损伤,并抑制Tf基因及蛋白表达。     

香烟烟雾提取物对A549细胞β防御素-2表达的影响

目的研究香烟烟雾提取物(cigarette smoke extract,CSE)对人肺腺上皮细胞A549β防御素-2(humanβ-defensin2,hBD-2)表达的影响。方法培养A549细胞,加或不加LPS(10μg/ml)情况下用5%CSE进行干预。用RT-PCR法和Western Blot法分别检测hBD-2mRNA和蛋白表达变化。结果LPS可以诱导A549细胞hBD-2的表达;5%CSE不影响A549细胞hBD-2的表达,但可抑制LPS的诱导作用。结论CSE可以抑制LPS诱导的肺腺上皮细胞hBD-2表达,这可能与CSE减弱呼吸道抵御外界微生物入侵能力,导致呼吸道感染及慢性阻塞性肺疾病有关。     

吸烟致大鼠肺组织蛋白质电泳图谱的改变

[目的]用双向电泳法测定烟草烟雾染毒大鼠肺组织差异蛋白的表达。[方法]雄性Wistar大鼠20只,平均体重(150±10)g,随机分为4组,每组5只。其中,3组为吸烟组,分别暴露于动式气体染毒装置进行烟草烟雾染毒1个月、2个月和4个月,每天2次,每次30min;另1组为不吸烟对照组,按常规饲养于动物房。分离不同剂量组大鼠肺组织,提取总蛋白,进行双向电泳测定。用ImageMaster2D Platinum软件对获得的蛋白图谱加以分析,寻找差异表达的蛋白质。[结果]病理切片显示吸烟组大鼠肺气肿改变明显,支气管炎症反应逐渐加重。双向电泳结果显示吸烟组大鼠肺组织总蛋白数增加,与对照组相比,有28个差异表达的蛋白点数,其中16个表达上调,12个表达下调。表达上调的蛋白点中,6个仅在吸烟组表达;下调的点中5个仅在对照组表达。[结论]双向电泳是一种有效的分离肺组织总蛋白的方法,烟草烟雾染毒可致大鼠发生肺气肿和支气管炎症反应,并引起大鼠肺组织蛋白质双向电泳图谱的改变。     

香烟烟雾对A549与A549-R细胞的氧化损伤

目的探讨hOGG1基因低表达对香烟烟雾作用下细胞氧化损伤效应的影响。方法不同浓度香烟烟雾暴露A549细胞和hOGG1基因低表达的A549-R细胞,MTT实验检测两种细胞存活率,彗星实验观察两种细胞DNA损伤,荧光法测定两种细胞内活性氧(ROS)含量,高效液相色谱-电化学法(HPLC-ECD)检测细胞基因组DNA中8-羟基脱氧鸟苷(8-OHdG)的含量。结果随着香烟烟雾暴露浓度的增加,两种细胞的存活率均下降,且A549-R细胞IC50显著小于A549细胞,差异有统计学意义(P<0.05);两种细胞ROS生成量与香烟烟雾浓度呈剂量-效应关系,当香烟烟雾浓度≥1.25支/升时,A549-R细胞ROS含量显著高于A549细胞(P<0.05);不同香烟烟雾浓度下,A549-R细胞拖尾率、尾长、OTM值均大于A549细胞,差异有统计学意义(P<0.05);A549细胞与A549-R细胞基因组DNA8-OHdG含量随香烟烟雾浓度的增加而升高,当香烟烟雾浓度为2.5支/升和5支/升时,A549-R细胞8-OHdG含量显著高于A549细胞(P<0.05)。结论香烟烟雾可导致A549细胞与A549-R细胞的氧化损伤,hOGG1基因低表达可增加A549-R细胞对香烟烟雾引起的氧化损伤的敏感性。     

二氧化硅对人呼吸道上皮细胞及巨噬细胞内IL-8和TNFα的诱导作用

[目的]探讨二氧化硅对人支气管上皮细胞（BEAS-2B）、Ⅱ型肺泡上皮细胞（A549）及巨噬细胞（THP-1）内白细胞介素-8（IL-8）和肿瘤坏死因子（TNFα）基因表达的诱导作用。[方法]将100μg/mL二氧化硅悬液与上述3种细胞分别温育4h,提取总RNA,用定量PCR技术测定生理盐水（对照组）与二氧化硅染毒组细胞内IL-8及TNFα mRNA表达水平。[结果]与对照组相比,在同一实验剂量及刺激时间（100μg／mL,4h）下,二氧化硅刺激可致THP-1内IL-8及TNFα mRNA表达水平分别升高至76．79倍及4．49倍;BEAS-2B内IL-8及TNFα mRNA表达水平分别升高至3．46倍及2．33倍;A549内IL-8及TNFα mRNA表达水平分别升高至1．64倍及1．52倍。[结论]二氧化硅可提高矽肺发病相关细胞因子IL-8及TNFα基因的表达水平。二氧化硅的上述作用,以对THP-1内IL-8 mRNA的诱导作用为最强。     

香烟烟雾暴露对大鼠睾丸组织雄激素结合蛋白mRNA及蛋白表达水平的影响

目的探讨香烟烟雾暴露对大鼠睾丸结构及睾丸组织雄激素结合蛋白(ABP)mRNA及蛋白表达水平的影响。方法清洁级成年雄性SD大鼠160只,随机分为对照组和低(10支)、中(20支)、高(30支)剂量染毒组,各组大鼠分别染毒2、4、6、8、12周,共16组,每组10只。实验组采用呼吸道静式染毒,每日1次,每次30 min。观察大鼠睾丸组织结构,采用RT-PCR及Western Blot法检测睾丸组织ABP mRNA及蛋白表达水平。结果染毒组大鼠睾丸病理切片可见,生精细胞排列紊乱疏松,支持细胞呈现空泡化。大鼠睾丸组织ABP mRNA和蛋白表达水平均随染毒剂量的增加而降低,染毒4周及12周后中、高剂量组ABP基因mRNA表达水平低于对照组,染毒6周后高剂量组ABP基因mRNA表达水平低于对照组,染毒4~8周后高剂量组ABP蛋白表达水平低于对照组,染毒12周后低、中、高剂量组ABP蛋白表达水平低于对照组,差异均有统计学意义(P0.05)。结论香烟烟雾暴露可引起雄性大鼠睾丸组织受损,且睾丸组织ABP基因mRNA及蛋白的表达水平下降。     

Tobacco smoke inhalation studies: a dosimetric comparison of different cigarette types

As part of a long-term inhalation bioassay study of cigarette smoke in rats, a detailed dosimetric comparison of three groups of rats exposed to smoke from different cigarette types was performed. Groups of 20 female F-344 rats were exposed, in Maddox-ORNL smoking machines, to 14C-dotriacontane-labeled smoke from three types of research cigarettes: high tar-low nicotine, low tar-high nicotine, and high tar-high nicotine. Analyses of lung tissues revealed similar deposition amounts and patterns of particulate matter for all three cigarette types even though the chamber smoke concentrations varied substantially. These results suggested that for rats exposed to different types of cigarette smoke, the amount of particulate material deposited may not be a function of concentration only. The authors conclude that when comparing cigarette smoke inhalation studies of different cigarette types, exposure parameters and smoke composition may both influence the amount of smoke inhaled and deposited in the lung and other organs.     

Synergistic effects of mineral fibres and cigarette smoke on the production of tumour necrosis factor by alveolar macrophages of rats.

The objective of this study was to evaluate the combined effects of mineral fibres and cigarette smoke on the production of tumour necrosis factor (TNF) by alveolar macrophages. Rats were exposed to cigarette smoke in vivo, and production of TNF by alveolar macrophages was measured in the presence of mineral fibres in vitro. For smoke exposure, rats were divided into two groups. Five were exposed to a daily concentration of 10 mg/m3 of cigarette smoke for an eight hour period, and five rats (controls) were not exposed to smoke. Bronchoalveolar lavage was performed after exposure to smoke and the recovered alveolar macrophages were incubated with either chrysotile or ceramic fibres on a microplate for 24 hours. Activity of TNF in the supernatant was determined by the L-929 fibroblast cell bioassay. When alveolar macrophages were not stimulated by mineral fibres, production of TNF by rats exposed to smoke and unexposed rats was essentially the same. When alveolar macrophages were stimulated in vitro by chrysotile or ceramic fibres, production of TNF by alveolar macrophages from rats exposed to smoke was higher than that by alveolar macrophages from unexposed rats. The findings suggest that cigarette smoke and mineral fibres have a synergistic effect on TNF production by alveolar macrophages.     

Dietary vitamin E and pulmonary biochemical responses of rats to cigarette smoking

The effect of dietary vitamin E on cellular susceptibility to cigarette smoking was studied in rats. Young male rats maintained on a basal vitamin E-deficient diet with or without 100 ppm vitamin E supplementation for 4 or 5 weeks were exposed to either sham or cigarette smoke for up to 7 days. Higher animal mortality rate was observed in the animals fed the vitamin E-deficient diet than in the supplemented group when they were subjected to acute levels of cigarette smoking. Relative to the respective sham groups, a greater alteration of biochemical parameters, such as reduced glutathione (GSH) and related enzymes, was found in the lungs of smoked rats fed the deficient diet than in the supplemented group. Animal lungs exhibited a greater biochemical response to whole smoke than the gaseous phase of smoke. The results suggest that the nutritional status of vitamin E may influence the cellular susceptibility of rats to cigarette smoking.     

Vitamin A depletion induced by cigarette smoke is associated with the development of emphysema in rats

We showed previously that vitamin A deficiency per se causes emphysema. Benzo(a)pyrene, a constituent in cigarette smoke, induces vitamin A depletion when administered to rats; therefore, we tested the hypothesis that cigarette smoke induces vitamin A depletion, which is associated with the development of emphysema. Male weanling rats were fed a purified AIN-93G diet and divided into two groups. The experimental group was exposed to cigarette smoke from 20 nonfiltered commercial cigarettes/d for 5 d/wk, whereas the control group was exposed to air. After 6 wk, tissues were collected for histological and biochemical analyses. Retinol levels were measured in serum, lung and liver. The trachea, lung and liver were examined for histological changes. Vitamin A levels decreased significantly in serum, lung and liver of smoke-treated rats. Histological examination revealed the presence of interstitial pneumonitis along with severe emphysema. There was a significant inverse relationship between vitamin A concentration in the lung and the severity of emphysema (r = -0.69 and P < 0.03). Detachment or hyperplasia (and metaplasia) of the tracheal epithelium and liver vacuole formation also were evident in the smoke-treated rats. The results of this research indicate that exposure to cigarette smoke induces vitamin A depletion in rats, which is associated with the development of emphysema.     

Coagulability of blood in rats exposed to cigarette smoke

Coagulability of the blood was studied in rats exposed acutely or chronically to cigarette smoke. No significant difference was found in plasma clotting time for control rats and rats chronically exposed to cigarette smoke when the measurement was made 24 hours after smoke exposure. When coagulation was measured during the period of smoke exposure, a significant shortening of plasma clotting time was observed but fibrinogen level, prothrombin time, activated partial thromboplastin time, and thrombin time were not altered. The duration of smoke-induced shortening of plasma clotting time was protracted in animals which had previously been exposed to smoke but was quickly rectified in animals which had no prior exposure to smoke. Furthermore, the degree of hypercoagulability during smoke exposure was more pronounced in old rats (24 months) than in young rats (2–7 months).     

beta-carotene attenuates the paradoxical effect of tobacco smoke on the mortality of rats after experimental myocardial infarction

The objective of this study was to investigate the effects of exposure to tobacco smoke (ETS) in rats that were or were not supplemented with dietary beta-carotene (BC), on ventricular remodeling and survival after myocardial infarction (MI). Rats (n = 189) were allocated to 4 groups: the control group, n = 45; group BC administered 500 mg/kg diet, n = 49, BC supplemented rats; group ETS, n = 55, rats exposed to tobacco smoke; and group BC+ETS, n = 40. Wistar rats weighing 100 g were administered one of the treatments until they weighed 200 to 250 g (approximately 5 wk). The ETS rats were exposed to cigarette smoke for 30 min 4 times/d, in a chamber connected to a smoking device. After reaching a weight of 200-250 g, rats were subjected to experimental MI (coronary artery occlusion) and mortality rates were determined over the next 105 d. In addition, echocardiographic, isolated heart, morphometrical, and biochemical studies were performed. Mortality data were tested using Kaplan-Meyer curves and other data by 2-way ANOVA. Survival rates were greater in the ETS group (58.2%) than in the control (33.3%) (P = 0.001) and BC+ETS rats (30.0%) (P = 0.007). The groups did not differ in the other comparisons. Left ventricular end-diastolic diameter normalized to body weight was greater and maximal systolic pressures were lower in the ETS groups than in non-ETS groups. Previous exposure to tobacco smoke induced a process of cardiac remodeling after MI. There is a paradoxical protector effect with tobacco smoke exposure, characterized by lower mortality, which is offset by BC supplementation.