用双向凝胶电泳分析低浓度烷化剂N-甲基-N′-硝基-N-亚硝胍引起的人羊膜FL细胞蛋白质的表达差异

目的 为了研究哺乳类细胞受到低浓度烷化剂攻击后蛋白表达谱的变化。方法 用双向凝胶电泳结合相应的2-DE分析软件比较烷化剂N-甲基-N′-硝基-N-亚硝基胍（MNNG）处理组和二甲基亚砜对照组的FL细胞的蛋白质组的表达差异。结果 MNNG处理后的FL细胞中检测到10个新出现的蛋白点，同时有5个蛋白点在MNNG处理后消失；有30个点在表达量上有显著变化，其中16个点在MNNG处理后表达升高，另14个点则表达量降低。结论 在低浓度烷化剂攻击的FL细胞中有一系列蛋白质表达水平的改变，提示这些发生改变的蛋白质可能参与了哺乳类细胞非定标性突变的发生。     

N-甲基-N′-硝基-N-亚硝基胍致大鼠气管上皮细胞体外转化及细胞遗传学改变

利用原代大鼠气管上皮(RTE)细胞研究N-甲基-N′-硝基-N-亚硝基胍(MNNG)的致转化作用，并对转化细胞进行细胞遗传学研究. 结果表明：增殖旺盛细胞(EGV)集落是RTE细胞体外转化最早期的特征，EGV转化频率与MNNG剂量呈明显的剂量反应关系. 从0.3 mg·L^-1 MNNG剂量组分离到2个EGV集落，经消化，传代形成了永生化的细胞系(RTES1,RTES2)，RTES1为多倍体核型，2号和7号染色体数目增加至4个且呈高频发生(100%); RTES2呈二倍体核型. 电镜结果证实转化细胞来自大鼠气管上皮组织. 以上结果提示，原代RTE细胞体外培养模型是研究致癌物定量及致癌机理的理想模型系统.     

N-甲基-N′-硝基-N-亚硝基胍诱发的vero细胞蛋白酪氨酸磷酸化改变

为了研究细胞信号转导通路在致突变物N-甲基-N′-硝基-N-亚硝基胍(MNNG)致非定标性突变作用中的地位,猴肾vero细胞在MNNG处理后0,6,12 h制备全细胞抽提液,用Western印迹法观察细胞蛋白质酪氨酸磷酸化的变化.结果发现0.2 μmol·L^-1 MNNG处理2.5 h后再经0 h和12 h,45 ku蛋白质酪氨酸磷酸化程度增强,经6 h时还有62 ku蛋白质酪氨酸磷酸化程度增强,1 mg·L^-1蛋白合成抑制剂环己米特(CHM)处理vero细胞12 h也可引起45 ku蛋白质酪氨酸磷酸化程度增强.提示MNNG处理可能诱发应激相关信号转导通路的激活.     

速激肽受体拮抗剂对白三烯C4诱导豚鼠气道收缩和微血管渗漏的作用

本实验探讨了内源性速激肽是否参与白三烯C₄(LTC₄)的气道效应. LTC₄(0.5 μg·kg^-1, iv)可增高豚鼠肺内压(IPP)和气道内依文思蓝渗出。速激肽NK-1受体拮抗剂CP-96345｛(2S, 3S)-顺式-2-( 二苯甲基)-N-［(2-甲氧苯)-甲基］-1-杂氮双环［2.2.2］辛烷-3-胺｝ 1 mg·kg^-1，iv，可减弱LTC₄诱导的依文思蓝渗出；NK-2受体拮抗剂SR-48968｛(S)-N-甲基-N-［4-(4-乙酰氨基-4-苯基哌啶)-2-(3,4-二氯苯基)丁基］苯甲酰胺｝，1 mg·kg^-1， iv，可抑制IPP的增高. 白三烯拮抗剂ONO-1078 (0.03 mg·kg^-1, iv)可阻断这两种反应. 结果说明内源性速激肽增强 LTC₄的气道作用，其中NK-1受体介导微血管渗漏，NK-2受体介导支气管收缩.     

吸烟人群细胞色素P4501A1和谷胱甘肽-S-转移酶M1的基因型

采用分子流行病学的方法,研究吸烟人群细胞色素P450 1A1基因(CYP 1A1)和谷胱甘肽-S-转移酶M1基因（GST M1）的多态性特征.吸烟人群与同性别及同年龄（年龄<2周岁）健康人群一对一配对. 提取血液基因组DNA. PCR扩增CYP 1A1 Exon 7位点片段和GST M1部分片段. 限制性酶切片段长度多态性（RELP）分析P450 1A1片段和GST M1片段多态性特点. 结果显示吸烟组人群中CYP 1A1 Exon 7 Val/Val型（mm型）人群数增高,并有统计学差异. 吸烟组人群中：GST M1缺失人群数略有升高;CYP 1A1 wm型人群数略有升高;CYP 1A1 wm型＋mm型人群数略有升高,但这些变化均无统计学意义.     

肿瘤坏死因子和一氧化氮在N-甲基-N-(3，4-亚甲二氧基苯甲酰)甲基-乙酰胺抗小鼠免疫性肝损伤中的作用

研究了N-甲基-N-（3，4-亚甲二氧基苯甲酰）甲基-乙酰胺（SY-640）的保肝作用及其机理. 给小鼠iv活卡介苗(BCG)12 d 后再iv脂多糖 (LPS)诱导小鼠血浆一氧化氮 (NO), 肿瘤坏死因子(TNF), 谷丙转氨酶(GPT), 谷草转氨酶 (GOT)剧烈升高及严重的肝损伤. 以SY-640给小鼠ig(每日一次，连续10 d)，显著降低BCG＋LPS 诱导的肝损伤小鼠血浆GPT，GOT和TNF水平的升高, 血浆NO水平的升高更加显著, 肝损伤减轻. 以单甲基精氨酸抑制NO的生成, SY-640的上述作用被抵消. SY-640对正常小鼠血浆NO，GPT，GOT水平无影响. 可见，SY-640 的保肝作用与其升高血浆NO, 降低血浆TNF水平有关.     

N-甲基-N′-硝基-N-亚硝基胍引起vero细胞基因表达改变及有关cDNA片段的初步鉴定

运用ｍＲＮＡ差异显示（ＤＤ－ＰＣＲ）技术，通过２６对锚定及任意引物组合显示基因的差异表达情况，分离了７个表达有差异的片段．其中３个片段的相关基因属于对Ｎ－甲基－Ｎ′－硝基－Ｎ－亚硝基胍（ＭＮＮＧ）处理的初级反应基因，２个属于次级反应基因．另有２个片段的差异表达仅在蛋白合成抑制剂环己亚胺（ＣＨＭ）合并ＭＮＮＧ处理时才出现．反向缝隙印迹杂交分析印证了２个片段在ＤＤ－ＰＣＲ中发现的改变，同时分析的本实验室分离的２５个片段中，８个片段的变化被验证与ＤＤ－ＰＣＲ中的改变一致．序列分析结果显示这些片段与许多基因有高同源性，其中包括一些参与信息传导的基因．     

环噻嗪对大鼠海马谷氨酸受体/NO/cGMP通路的调节

通过活体微透析的方法研究了环噻嗪对大鼠海马谷氨酸受体/NO/cGMP通路的影响. 局部灌流α- 氨基羟甲基异噁唑丙酸(AMPA)受体脱敏阻断剂环噻嗪能引起细胞外cGMP水平的提高. 环噻嗪的这种作用能够被NO合酶抑制剂N-硝基-L-精氨酸(L-NNA)或选择性的可溶性鸟苷酸环化酶抑制剂1H-［1，2，4］噁二唑［4，3-a］喹喔啉-1-酮(ODQ)所阻断. 在环噻嗪灌流过程中，大鼠呈现明显的痉挛前的行为变化--湿狗样反应(WDS). 由环噻嗪引起的cGMP增加和WDS反应能够被N-甲基-D-天冬氨酸(NMDA)受体通道阻断剂甲基二苯并环庚烯亚胺(MK-801)或镁离子所阻断. AMPA受体拮抗剂6，7-二硝基喹喔啉-2，3-二酮(DNQX)和2，3-二羟基-6-硝基-7-氨磺酰基 苯并(f)- 喹喔啉(NBQX)可拮抗WDS反应，但不能阻断环噻嗪引起的cGMP反应. 这种结果表明：（1）在海马内与NO-cGMP通路有关的AMPA受体由于内源性谷氨酸的存在保持部分脱敏状态.（2）环噻嗪对AMPA受体脱敏的阻断作用可导致内源性NMDA受体的激活.     

一个参与N-甲基-N′-硝基-N-亚硝基胍诱发的遗传不稳定的cDNA片段的分离筛选

为研究甲基硝基亚硝基胍（MNNG）诱发猴肾vero细胞遗传不稳定的机理，采用mRNA差异显示分离到的MNNG处理后表达发生改变的表达序列标识（EST）相关基因进行功能筛选. 利用反义核酸阻断技术阻断其相应基因的表达，以筛选分离参与非定标性突变形成的cDNA片段. 现筛选到10号片段，其相关基因的表达被阻断后，MNNG诱发的vero细胞非定标突变率下降至自发突变水平，提示10号片段相关的基因是非定标突变发生的正相关基因.     

外消旋普罗帕酮在经诱导的大鼠肝微粒体中N-去丙基化的立体选择性

采用对照及β-萘黄酮(β-NF)或地塞米松(Dex)诱导的大鼠肝微粒体,应用GITC柱前衍生化,反相高效液相色谱法研究了消旋普罗帕酮〔(R/S)-PPF〕体外代谢的立体选择性. 实验结果表明,在Dex,β-NF诱导的微粒体中有N-去丙基普罗帕酮生成。在β-NF,Dex预处理组,(R/S)-PPF低浓度时的经肝微粒体代谢具有立体选择性,R(-)对映体的清除大于S(+)对映体,高浓度时的代谢无立体选择性. R(-)对映体的K_m值显著低于S(+)对映体,而V_max值无显著性差异. 在Dex预处理组中的立体选择性大于β-NF组. 在对照组中代谢无立体选择性,且K_m,V_max值均小于β-NF,Dex预 处理组。结果提示,CYP1A,CYP3A4亚族对普罗帕酮(PPF)的N-去丙基化有贡献. (R/S)-PPF的N-去丙基化具有浓度依赖性的立体选择性.     

一个参与N-甲基-N′-硝基-N-亚硝基胍诱发的遗传不稳定的cDNA片段的分离筛选

为研究甲基硝基亚硝基胍 ( MNNG)诱发猴肾vero细胞遗传不稳定的机理 ,采用 m RNA差异显示分离到的 MNNG处理后表达发生改变的表达序列标识 ( EST)相关基因进行功能筛选 .利用反义核酸阻断技术阻断其相应基因的表达 ,以筛选分离参与非定标性突变形成的 c DNA片段 .现筛选到 1 0号片段 ,其相关基因的表达被阻断后 ,MNNG诱发的 vero细胞非定标突变率下降至自发突变水平 ,提示 1 0号片段相关的基因是非定标突变发生的正相关基因 .     

Microarray analysis of gene expression patterns in human proximal tubule cells over a short and long time course of cadmium exposure

Numerous studies showed that renal proximal tubules cells are the cell type critically affected by chronic exposure to cadmium (Cd(2+)). The aim of the present study was to apply global gene expression technology and a human renal epithelial cell culture model (HPT) to determine whether time of exposure to Cd(2+) exerts a major influence on the resulting pattern of global gene expression. HPT cells were exposed to Cd(2+) for a short, 1-d, period of exposure (9, 27, and 45 μM) versus a longer, 13-d, period (4.5, 9, and 27 μM), with the hypothesis being that the stress response of the cells would be more active during the short time of exposure. The results showed that the differential expression of genes was very extensive for HPT cells exposed to Cd(2+) for 1 d, with more than 1848 genes displaying alterations compared to control and with the major categories of genes being involved in stress responses; cell death; checkpoint arrest, DNA repair, and the cell cycle; inflammatory responses; and cell adhesion, motion and differentiation. In contrast, HPT cells exposed to Cd(2+) for 13 d showed 923 genes to be differentially expressed, with a marked reduction in the number of differentially expressed stress response genes and a significant increase in the number of genes involved in development and differentiation. There were 387 differentially expressed genes common to both times of exposure. Data suggest that unless one is actively seeking to study the acute stress response, global gene expression technology should not be applied within an early time course of toxicant exposure.     

Gene expression profile changes induced by acute toxicity of benzo[a]pyrene in marine medaka

Differential gene expression profiling was carried out using cDNA microarray hybridization on hepatic tissue from marine medaka (Oryzias javanicus) after exposure to benzo[a]pyrene (BaP), a representative polycyclic aromatic hydrocarbon classified as a persistent organic pollutant. Forty-one differentially expressed candidate genes were identified; 18 were induced and 23 were repressed (P/0.05). The genes were assembled into 18 groups based mainly on the Eukaryotic Orthologous Groups classification. These differentially expressed gene candidates could have great potential as molecular biomarkers for identifying environmental stressors and prognosis for the biological effects of BaP. The candidate genes isolated in this study were grouped into endocrine disruption, cardiovascular disease, tumorigenesis, immune response, detoxification, energy production and conversion, and other biological responses. Our results could allow future studies to assess the molecular mechanisms of BaP toxicity and to develop a systems biology approach to environmental stress biology.     

Differential gene expression profiling in iprobenfos-exposed marine medaka by heterologous microarray hybridization

Differential gene expression profiling was performed in the hepatic tissue of marine medaka fish (Oryzias javanicus) after exposure to an organophosphorous pesticide (OPP), Iprobenfos (IBP), a widely used pesticide in agri- and fish-culture, by heterologous hybridization using a medaka cDNA microarray. Fifty differentially expressed candidate genes, of which 10 induced and 40 repressed (P<0.01), were identified. The genes were assembled into 19 groups mainly based on Eukaryotic Othologous Groups (KOG) classification. These isolated gene candidates were differentially expressed and therefore have great potential as molecular biomarkers for the identification of environmental stressors. The results obtained in this study will allow future studies to assess the molecular mechanisms of IBP toxicity and the development of a systems biology approach to the stress biology of organophosphorous pesticide.     

Heterologous microarray hybridization used for differential gene expression profiling in Arochlor 1260-exposed marine medaka

Differential gene expression profiling was performed by heterologous hybridization using a medaka cDNA microarray on the hepatic tissue of the marine medaka (Oryzias javanicus) after exposure to Arochlor 1260, a polychlorinated biphenyl (PCB), which is classified as a persistent organic pollutant. Twenty-eight differentially expressed candidate genes were identified; one was induced and 27 were repressed (P<0.01). The genes were assembled into 10 groups based mainly on the Eukaryotic Othologous Groups classification. These isolated gene candidates were differentially expressed and therefore have great potential as molecular biomarkers for identifying environmental stressors. The results obtained in this study will allow future studies to assess the molecular mechanisms of Arochlor 1260 toxicity and to develop a systems biology approach to PCB stress biology.     

甲基亚硝基胍致小鼠腭裂的分子机制

目的探讨小鼠腭裂发生的分子机制。方法以甲基亚硝基胍(MNNG)为受试物诱导腭裂动物模型,采用抑制消减杂交技术从全基因组水平上对MNNG致腭裂相关差异表达基因进行筛选。结果得到MNNG逆向表达片断27个,正向差异表达片断14个。经测序和GeneBank比对后得到已知基因9条,余者为未知功能基因或未知基因。结论Gpc3可能通过调节腭发生过程中某些基因干扰间充质细胞正常增殖;Ptprs的表达抑制影响了某种腭突发生中细胞信号转导通路;腱生蛋白的抑制表达可能使细胞粘连力的去除失败或者由于基质中MMPs的表达降低而使腭中缝消除受阻;抑或由EgfrPtprsTnC协同作用而导致腭裂发生。还可能影响细胞对营养物质的摄取而引起Rps25的上调诱发过度的细胞凋亡而在腭裂发生中发挥作用。     

Identification of Differentially Expressed Genes with Multivariate Outlier Analysis

Abstract

DNA microarray offers a powerful and effective technology to monitor the changes in the gene expression levels for thousands of genes simultaneously. It is being widely applied to explore the quantitative alternation in gene regulation in response to a variety of aspects including diseases and exposure of toxicant. A common task in analyzing microarray data is to identify the differentially expressed genes under two different experimental conditions. Because of the large number of genes and small number of arrays, and higher signal-noise ratio in microarray data, many traditional approaches seem improper. In this paper, a multivariate mixture model is applied to model the expression level of replicated arrays, considering the differentially expressed genes as the outliers of the expression data. In order to detect the outliers of the multivariate mixture model, an effective and robust statistical method is first applied to microarray analysis. This method is based on the analysis of kurtosis coefficient (KC) of the projected multivariate data arising from a mixture model so as to identify the outliers. We utilize the multivariate KC algorithm to our microarray experiment with the control and toxic treatment. After the processing of data, the differential genes are successfully identified from 1824 genes on the UCLA M07 microarray chip. We also use the RT-PCR method and two robust statistical methods, minimum covariance determinant (MCD) and minimum volume ellipsoid (MVE), to verify the expression level of outlier genes identified by KC algorithm. We conclude that the robust multivariate tool is practical and effective for the detection of differentially expressed genes.     

The kinetics of transcriptomic changes induced by cigarette smoke in rat lungs reveals a specific program of defense, inflammation, and circadian clock gene expression.

Gene expression profiling in animal models exposed to cigarette mainstream smoke (CS) shapes up as a promising tool for investigating the molecular mechanisms involved in the onset and development of CS-related disease and may aid in the identification of disease candidate genes. Here we report on differential gene expression in lungs of rats exposed for 2, 7, and 13 weeks to 300 and 600 microg total particulate matter/l CS with sacrifice 2, 6, or 20 h after the last exposure. Regarding antioxidant and xenobiotic-metabolizing (phase I/II) enzymes, a stereotypic, mostly transient, expression pattern of differentially expressed genes was observed after each exposure period. The expression patterns were generally dose dependent for antioxidant and phase II genes and not dose dependent for phase I genes at the CS concentrations tested. However, with increasing length of exposure, there was a distinct, mostly sustained and dose-sensitive, expression of genes implicated in innate and adaptive immune responses, clearly pointing to an emerging inflammatory response. Notably, this inflammatory response included the expression of lung disease-related genes not yet linked to CS exposure, such as galectin-3, arginase 1, and chitinase, as well as genes encoding proteolytic enzymes. Finally, our experiments also revealed a CS exposure-dependent shift in the cyclical expression of genes involved in controlling the circadian rhythm. Altogether, these results provide further insight into the molecular mechanisms of CS-dependent disease onset and development and thus may also be useful for defining CS-specific molecular biomarkers of disease.