4-（methylnitrosamino）-l-（3-pyridyl） -1-butanone Induces Circulating MicroRNA Deregulation in Early Lung Carcinogenesis

Objective To study the alteration of circulating microRNAs in 4-（methylnitrosamino）-l-（3-pyridyl） -l-butanone （NNK）-induced early stage lung carcinogenesis. Methods A lung cancer model of male F344 rats was induced with systemic NNK and levels of 8 lung cancer-associated miRNAs in whole blood and serum of rats were measured by quantitative RT-PCR of each at weeks 2, 5, 20, and 20 following NNK treatment. Results No lung cancer was detected in control group and NNK treatment group at week 20 following NNK treatment. The levels of some circulating miRNAs were significantly higher in NNK treatment group than in control group. The miR-210 was down-regulated and the miR-206 was up-regulated in NNK treatment group. The expression level of circulating miRNAs changed from week 1 to week 20 following NNK treatment. Conclusion The expression level of circulating miRNAs is related to NNK-induced early stage lung carcinogenesis in rats and can therefore serve as its potential indicator.     

青藤碱对肺癌细胞A549中COX2、α7nAChR和A2A表达的影响

【目的】观察青藤碱(sinomenine,SIN)对环氧化酶2(COX2)、α7烟碱型乙酰胆能受体(α7nAChR)和腺苷受体(A2A)表达变化的影响,探讨青藤碱对A549细胞增殖的抑制作用及机制。【方法】采用四甲基偶氮唑盐微量酶反应比色(MTT)法检测青藤碱和4-甲基亚硝胺基-1-3-吡啶基-1-丁酮(NNK)对A549细胞增殖的影响;细胞划痕实验观察青藤碱和NNK对A549细胞迁移的影响;Western blot法观察青藤碱和NNK对A549细胞COX2蛋白表达的影响;RT-PCR和Western blot法观察青藤碱和NNK对A549细胞α7n ACh R、A2A表达的影响。【结果】NNK能促进增殖和迁移,青藤碱能抑制A549细胞增殖和迁移;NNK组COX2蛋白质水平增加,青藤碱组COX2蛋白质水平下降;NNK组α7n ACh R、A2A的表达增加,青藤碱组α7n ACh R、A2A表达下降。【结论】青藤碱能通过抑制COX2蛋白表达发挥抗A549细胞增殖和迁移作用;青藤碱对α7n ACh R和A2A受体的表达均有抑制作用。     

过氧化物酶1对人肝癌细胞HepG2细胞皮下移植瘤生长的影响

目的  探讨过氧化物酶1（Prx 1）在肝癌形成和恶性进展中的作用。方法  蛋白质印迹法（Western blot）检测远癌肝组织和近癌肝组织中Prx 1、pAkt及Akt表达差异。肝细胞饥饿培养24 h后,10 ng/ml表皮生长因子（EGF）处理10 min,提取总蛋白,Western blot检测Prx 1、pAkt、不同位点磷酸化表皮生长因子受体（EGFR）和总EGFR。Western blot检测检测未处理组、转染无义shRNA组及转染Prx 1 shRNA组HepG2细胞中Prx 1蛋白的表达。DCFH-DA法检测3种HepG2细胞中活性氧簇总量。转染无义shRNA组及转染Prx 1 shRNA组HepG2细胞皮下注射至SCID小鼠,注射后每周测量1次皮下肿瘤体积。Western blot检测两组小鼠皮下肿瘤组织中Prx 1和pAkt的表达情况。结果  与正常肝细胞比较,Prx 1在肝转化细胞中表达升高。Prx 1增强了EGF介导的EGFR和Akt激活。Prx 1表达沉默抑制了HepG2体内成瘤能力。减少HepG2皮下移植瘤的肝转移。Prx 1表达沉默还抑制了Akt体内激活。结论  Prx 1具有调控人肝细胞中EGFR介导信号通路的潜在作用。Prx 1能够通过EGFR-Akt信号通路介导细胞正常至异常转化、肿瘤形成及转移

     

Peroxiredoxin 1 蛋白表达与肾积水发生的相关性

目的：研究Peroxiredoxin 1（Prx1）蛋白在2,3,7,8-四氯二苯并二噁英（2,3,7,8-tetrachlorodibenzo-p—dioxin,TCDD）所致胎鼠。肾积水形成过程中的作用．方法：以TCDD诱导建立胎鼠肾积水动物模型,观察胎鼠肾脏、输尿管的组织学改变和超微结构改变,用WesternBlot和免疫组化检测胎鼠肾脏、输尿管Prxl蛋白的表达情况．结果：利用TCDD能成功诱导建立胎鼠。肾积水动物模型．肾积水胎鼠输尿管黏膜上皮增生,输尿管管腔狭窄．WestemBlot表明实验组胎鼠上尿路Prxl蛋白表达比对照组明显增强,免疫组化示实验组胎鼠输尿管黏膜上皮Prxl蛋白表达为阳性,而对照组呈阴性．结论：TCDD所致胎鼠输尿管黏膜上皮Prxl蛋白高表达可能与肾积水发生相关．     

Peroxiredoxin 1与EMT相关蛋白在胰腺癌组织中的表达及其临床意义

目的：探讨过氧化物还原酶1( Prx1)与上皮细胞间质转化( EMT)相关蛋白在胰腺癌组织中的表达及其临床意义。方法采用免疫组化法分别检测 Prx1、E-钙黏素( E-cadherin)、波形蛋白( Vimentin)在66例胰腺癌组织和11例正常胰腺组织中的表达,并分析三者之间的关系及与临床病理因素间的关系。结果 Prx1、E-cadherin和Vimentin分别在66例胰腺癌组织中的阳性表达率为69．70%(46/66)、40．91%(27/66)、56．06%(37/66);Prx1、E-cadherin和Vim-entin分别在11例正常胰腺组织中的阳性表达率为18．18%(2/11)、90．90%(10/11)、18．18%(2/11)。 Prx1、E-cadherin和Vimentin在胰腺癌组织中的表达均与TNM分期、淋巴结转移和肿瘤分化程度显著相关(P<0．05),而与性别、年龄、肿瘤大小、病变部位均无关;Prx1与神经侵犯显著相关( P<0．05),而E-cadherin和Vimentin与神经侵犯无关。 Prx1的表达与Vimentin表达呈正相关(P<0．05),Prx1和Vimentin的表达均与 E-cadherin表达呈负相关( P<0．05)。结论Prx1与EMT相关蛋白在胰腺癌组织中的表达呈显著相关性,并与其侵袭转移能力相关,提示Prx1的高表达可能为预测胰腺癌患者预后不良的辅助指标之一。     

人支气管上皮细胞恶性转化过程中染色体畸变的研究

目的:研究在烟草特异性亚硝胺(NNK)诱发人支气管上皮细胞(BEAS-2B)恶性转化过程中染色体畸变,探讨其在吸烟高危人群罹患肺癌预警及普查方面的意义.方法用NNK诱发BEAS-2B细胞(BEAS-2BNNK)恶性转化,并在此过程中用常规中期染色体分析方法,动态观察染色体畸变的情况.结果:1.NNK诱发BEAS-2B细胞恶性转化模型的建立①BEAS-2BNNK(实验组)第5代细胞血清抗性显著增强.②第15代BEAS-2BNNK细胞具有锚着独立性生长特性.③第20代BEAS-2BNNK细胞超微结构出现明显异型性.④第25代BEAS-2BNNK细胞在裸鼠体内成瘤,病理为高分化鳞形细胞癌.2.染色体畸变①BEAS-2B(对照组)二倍体细胞占细胞总数的91%～97%,传代后核型稳定;从第5代开始BEAS-2BNNK细胞即逐渐失去正常核型,随着传代次数增加,二倍体细胞比例从83%递减至54%,异倍体及多倍体细胞呈递增趋势,较对照组明显增高.②各代BEAS-2B细胞结构畸变发生率为2%～4%;除第5代BEAS-2BNNK细胞结构畸变总频率(11%)明显高于BEAS-2B细胞外,其余各代细胞与BEAS-2B细胞相近.结论:NNK能成功诱发人支气管上皮细胞(BEAS-2B)细胞恶性转化,为进一步探讨肺癌发生机制、尤其是吸烟致肺癌发生机制提供了理想模型.染色体的异倍性和多倍性在NNK诱发BEAS-2B细胞转化成肺癌过程中可能属早期事件,有望成为吸烟高危人群罹患肺癌的预警及普查指标之一.     

过氧化物酶1,6和GFAP在人脑星形胶质细胞瘤中的表达及临床意义

目的确定星形胶质细胞瘤中过氧化物酶1(Prx1)、过氧化物酶6(Prx6)和胶质纤维酸性蛋白(GFAP)的表达情况,探讨其表达水平与星形胶质细胞瘤恶性程度的关系。方法采用Western blot、RT-PCR和免疫组化检测52例星形胶质细胞瘤(Ⅱ级23例、Ⅲ级15例、Ⅳ级14例)和12例正常脑组织标本中Prx1、Prx6和GFAP的表达情况。结果 Prx1、Prx6在正常脑组织、Ⅱ级、Ⅲ级、Ⅳ级星形胶质细胞瘤中蛋白和mRNA的表达水平逐渐升高,具有统计学意义(P<0.05);GFAP在Ⅲ、Ⅳ级星形胶质细胞瘤中蛋白和mRNA的表达水平低于Ⅱ级星形胶质细胞瘤和正常脑组织(P<0.05)。结论 Prx1、Prx6和GFAP可能是临床评价星形胶质细胞瘤恶性程度和侵袭性的潜在生物标志物,可作为星形胶质细胞瘤生物治疗的潜在靶分子。     

化学致癌物体外转化大鼠气管上皮细胞的研究

作者建立了大鼠气管上皮(rat tracheal epithelial,RTE)细胞的单层原代培养和RTE细胞体外转化实验,比较有滋养层和无滋养层RTE细胞转化系统的实验结果。首次证明烟草中的特殊亚硝胺——甲基亚硝基吡啶基丁酮(NNK)对RTE细胞有体外转化作用。在甲基硝基亚硝基胍(MNNG)和NNK诱导转化实验中,有滋养层和无滋养层体外转化系统得到的结果一致,而后者在RTE细胞体外转化应用中更为敏感而方便。     

Hemoglobin adducts as biomarkers of exposure to and metabolic activation of carcinogenic tobacco-specific nitrosamines

The carcinogenic tobacco-specific nitrosamines N'-nitrosonornicotine (NNN) and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) form hemoglobin adducts in laboratory animals and humans. These adducts release 4-hydroxy-1-(3-pyridyl)-1-butanone (HPB) upon mild base hydrolysis. HPB released from human hemoglobin can be quantified by gas chromatography-mass spectrometry. It is the only available biochemical marker for determination of exposure to, and metabolic activation of, carcinogens present only in tobacco. Levels of HPB were highest in snuff-dippers, followed by smokers and nonsmokers. Large interindividual variations in adduct levels were observed. The relationship between HPB levels in globin and DNA of rats treated with NNK has been investigated in order to aid in interpretation of the data from humans. These studies have provided the initial database for understanding the metabolic activation of tobacco-specific nitrosamines in humans.     

Cotinine effects on bile flow and biliary NNK elimination

Nicotine and its major metabolite cotinine inhibit alpha-hydroxylation of the tobacco-specific nitrosamine 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) suggesting that an alternative pathway of NNK metabolism and elimination, biliary excretion of the O-glucuronide of 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol (NNAL-Gluc) may be enhanced. To verify the possible role of cotinine on biliary elimination of NNK and its metabolites, bile duct cannulated rats were administered a single i.p. dose of 50 mg/kg [56sup;-3 H]-NNK with or without i.p. co-administration of 5 mg/kg cotinine or nicotine. Cotinine significantly reduced cumulative bile flow and biliary elimination of NNK-derived radioactivity within six hours to 42 and 27 percent, respectively. The pattern of NNK metabolites in bile was unchanged. Nicotine had a similar inhibitory effect on bile flow. This result constitutes the first experimental evidence that cotinine inhibits bile flow. In rats, biliary elimination of NNK is reduced accordingly which may lead to an increased carcinogen burden in the body. In humans, inhibition of bile flow by tobacco alkaloids may contribute to the appetite suppressing effect of tobacco products.     

热休克蛋白在实验性大鼠脑死亡肾中的表达

目的研究热休克蛋白(HO-1、HSP27和HSP70)在脑死亡大鼠肾中的表达。方法将气囊导管插入F344雄鼠大脑硬膜外慢慢充气,诱导脑死亡,未处理组做对照组,4 h后处死大鼠,采用RT-PCR、蛋白印迹和免疫组化检测肾脏HO-1、HSP27和HSP70的表达。结果RT-PCR显示HO-1(P<0.01)和HSP70(P<0.01)基因编码表达增强。蛋白印迹也显示HO-1蛋白水平增加(P<0.01),但HSP70蛋白表达无明显变化。免疫组化显示HO-1蛋白在肾皮质表达增强。HSP27在脑死亡供体肾的表达无变化。结论脑死亡引起的肾应激可诱导细胞保护基因HO-1和HSP70的表达。     

Entamoeba histolytica modulates TNF-alpha, IL-1 alpha/beta and c-fos gene expression in macrophages.

E. histolytica infections induce a state of transient suppression of cell-mediated immunity. As macrophages are involved in host defense in amebiasis, we determined whether soluble amebic lysates (Eh) can modulate TNF-alpha, IL-1 alpha/beta and c-fos gene expression in naive bone marrow-derived macrophages (BM delta). By Northern analysis, the RNA production of these genes after 0, 0.5, 1 and 3 h exposure to Eh was determined and compared to lipopolysaccharide (LPS) stimulation. In response to Eh, TNF-alpha mRNA was increased two fold while IL-1 alpha/beta RNA levels were increased 6- and 19-fold, respectively. Pretreatment of BM delta with H7, a PKC inhibitor, abrogated Eh induced TNF-delta gene expression and reduced IL-1 alpha/beta gene expression 3.5- to 4-fold over control levels. We conclude that E. histolytica stimulates BM delta to induce TNF-alpha gene expression through a PKC-dependent pathway and IL-1 alpha/beta gene expression partially through PKC and another yet undetermined pathway(s).     

慢性应激对大鼠下丘脑CRHR1基因启动子区域DNA甲基化的作用

目的:研究慢性应激所致的动物下丘脑促肾上腺皮质激素释放激素受体1(CRHR1)表达和基因启动子区域甲基化状态关系。方法:将大鼠随机分为抑郁组和对照组各9只,抑郁组进行21d的慢性不可预见性温和应激。进行行为学观察,用荧光定量PCR、蛋白免疫印迹和亚硫酸氢盐修饰后测序分别检测mRNA及蛋白质表达和DNA甲基化。结果:抑郁组行为学有所改变,mRNA及蛋白质表达升高,均与对照组有显著差异,而DNA甲基化没有明显不同。结论:慢性应激后,CRHR1表达上调,但其非由DNA甲基化机制调控。     

高糖增加大鼠肺微血管内皮细胞基质金属蛋白酶10基因表达

目的 研究葡萄糖对培养血管内皮细胞表达的影响。方法 大鼠肺微内皮细胞在高浓度糖(高糖)和正常浓度糖培养基中培养24h，作基因芯片分析。结果 在超过4000个基因芯片显示的基因中，高糖引起509个基因表达增加，961基因表达减少。其中细胞基质金属蛋白酶(matrix metalloproteinase，MMP)10基因表达增加6．06倍，Wilcoxon配对符号秩检验P=0．0001。结论 MMP10在高糖环境下培养的大鼠肺内皮细胞表达增加，可能对糖尿病慢性并发症的形成过程起一定作用。     

全反式维甲酸通过内质网应激诱导N-乙酰氨基葡萄糖基转移酶V受阻的人肝癌SMMC-7721细胞凋亡

目的初步探讨全反式维甲酸（all-trans-retinoic acid，ATRA）诱导N-乙酰氨基葡萄糖基转移酶V（N—acetylglucosaminyltransferase V，GnT—V）表达受阻的人肝癌SMMC-7721细胞（AsGnT—V／SMMC-7721）发生凋亡与细胞中内质网应激的关系。方法利用RT-PCR检测ATRA处理前后AsGnT-V／SMMC-7721细胞中内质网应激关键分子GRP78（glucose regulated protein 78）、XBP1（X box binding protein 1）等的变化。并用Western blot检测ATRA处理前后AsGnT—V／SMMC-7721细胞中内质网应激相关凋亡途径中的重要分子CHOP（C／EBP homologus protein）、caspase-9、caspase-12以及caspase-3的变化。结果GRP78和XBP1的变化表明经ATRA处理后AsGnT-V／SMMC-7721细胞的内质网应激加剧了，而与内质网应激相关的凋亡分子CHOP、caspase-9、caspase-12以及caspase-3都发生了激活。结论ATRA诱导AsGnT—V／SMMC-7721细胞发生的凋亡与内质网应激有关。     

Toxicity and Carcinogenicity of Ozone in Combination with 4-（N-methyl-N-nitrosamino）-1-（3-pyridyl）-1-butanone and Dibutyl Phthalate in B6C3F1 Mice for 16 and 32 Weeks

Objective To evaluate the toxic and carcinogenic potential of ozone alone or in combination with 4-（N-methyl-N-nitrosamino）-1-（3-pyridyl）-1-butanone （NNK） and/or dibutyl phthalate （DBP）. Methods Male and female B6C3F1 mice were exposed, through inhalation, intravenous administration and diet, to 0.5 ppm of ozone, 1.0 mg/kg of NNK and 5000 ppm of DBP, individually and in combination for 16 and 32 weeks. Results No treatment-related death was seen, but significant differences in body and organ weights between control and treated mice were observed during the study. No incidence of lung tumor incidence was recorded in mice exposed to either ozone alone or combined treatment. Oviductal carcinomas were observed in female mice exposed to ozone or DBP alone for 16 weeks and ozone in combination with NNK and DBP for 32 weeks. Conclusion Although ozone alone and in conjunction with NNK and/or DBP does not induce lung cancer under our experimental conditions, they induce oviductal carcinomas in B6C3F1 mice.     

过氧化物还原酶4在大肠癌组织中的表达及其意义

目的：研究过氧化物还原酶4（peroxiredoxin 4,Prx4）在大肠癌组织中的表达及其临床意义。方法：采用免疫组织化学法检测59例手术切除大肠癌及26例癌旁正常组织中Prx4蛋白的表达情况,并分析其与患者临床病理特征的关系。结果：大肠癌组织中Prx4蛋白阳性率明显高于相应的癌旁组织（P〈0.05）;Prx4蛋白的表达强度与大肠癌的肿瘤浸润深度、淋巴转移、Dukes分期及生存期均有相关性（P〈0.05）。结论：Prx4在大肠癌组织中表达水平高于癌旁正常组织,并与是否伴有淋巴转移、肿瘤浸润深度和Dukes分期有关,提示Prx4与大肠癌的发生发展有关。     

血管紧张素1-7降低脂肪细胞氧化应激增加脂联素表达

目的 研究血管紧张素（1-7）[Ang-（1-7）]/Mas轴对于高糖培养脂肪细胞氧化应激及脂联素表达的作用.方法用前脂肪细胞株3T3L1诱导分化成成熟细胞,分别在培养基中加入外源性的Ang-（1-7） 10-9mol/L,Mas受体抑制剂A779 10-6mol/L,同时加入Ang-（1-7）及A779.分别用DHE荧光探针通过流式细胞术及用NBT染色法检测脂肪细胞内活性氧（ROS）含量.实时定量PCR方法检测脂联素及炎性反应因子的表达.无血清培养基培养脂肪细胞2 h后分别加入葡萄糖氧化酶（GO）50 mU/mL、100 mU/mL作用12 h建立氧化应激模型,研究Ang-（1-7）对于脂肪细胞氧化应激的作用及对脂联素表达的影响.结果 ①Ang-（1-7）通过Mas受体降低脂肪细胞活性氧的产生.Mas受体抑制剂A779可逆转此作用.②构建氧化应激模型,脂肪细胞中的脂联素表达水平随着葡萄糖氧化酶浓度的增高而减少.③加入Ang-（1-7）可以逆转氧化应激导致的脂联素表达降低,抑制剂A779可以对抗Ang-（1-7）的这一作用.④Ang-（1-7）对于白细胞介素-6（IL-6）和肿瘤坏死因子-α（TNF-α）的mRNA表达水平没有影响.结论 Ang-（1-7）/Mas轴可以减少氧化应激产生的活性氧水平.Ang-（1-7）/Mas轴对氧化应激的保护作用可以增加抗胰岛素抵抗因子脂联素的表达.