牛分枝杆菌感染巨噬细胞（THP-1）全基因表达谱变化的研究

目的分析可用于牛结核病免疫学诊断的候选靶标,同时初步探讨由牛分枝杆菌（Mycobacterium bovis）引起人结核病的免疫学发病机制。方法牛分枝杆菌 (AF 2212/97)和结核分枝杆菌（Mycobacterium tuberculosis(H₃₇Rv)）感染人的模式巨噬细胞（THP-1）,采用含有22000个基因全长的基因芯片分析感染72h后的THP-1细胞全基因组的在转录水平的表达谱变化。在确定所分析的芯片的特异性和稳定性均达到要求的基础上,通过GO显著性统计分析,研究表达变化的基因所对应蛋白的分子功能,生物过程以及细胞组分;进一步采用Pathway显著性统计分析,研究表达差异蛋白之间的相互作用。结果感染H₃₇Rv后的THP-1细胞,存在290个差异基因表达,其中170个上调表达,120个下调表达,与以往的结果类似。牛分枝杆菌感染的THP-1细胞中有1899(8.6%)个基因差异表达,其中1075个基因被上调表达(56.6%),其他43.4%被下调。1899个基因中1674个在GenBank有记录,263个是新基因,,基质金属蛋白酶12(H200000442)被上调达60.4921倍;下调幅度最大的蛋白丝氨酸/半胱氨酸抑制酶(H200006177)被下调6.29倍。采用GO分析发现,这些基因对应的多为炎症因子蛋白、凋亡蛋白、细胞外基质蛋白、T细胞受体信号通路相关蛋白等。其中与炎症相关的的蛋白有47个,如TNF、IL8、CXCL3、CCL8、CCL7、IFNK、IL-26、CCL28等。随后的Pathway分析差异表达基因对应的2014个蛋白,展示了一系列的引起结核病发病的重要信号传导通路。结论基因表达谱研究可为牛结核病发病机制及其诊断研究提供大量有益的生物学信息。     

An international standardization programme towards the application of gene expression profiling in routine leukaemia diagnostics: the Microarray Innovations in LEukemia study prephase

Gene expression profiling has the potential to enhance current methods for the diagnosis of haematological malignancies. Here, we present data on 204 analyses from an international standardization programme that was conducted in 11 laboratories as a prephase to the Microarray Innovations in LEukemia (MILE) study. Each laboratory prepared two cell line samples, together with three replicate leukaemia patient lysates in two distinct stages: (i) a 5-d course of protocol training, and (ii) independent proficiency testing. Unsupervised, supervised, and r(2) correlation analyses demonstrated that microarray analysis can be performed with remarkably high intra-laboratory reproducibility and with comparable quality and reliability.     

基因芯片技术对哮喘病人相关基因的筛选和分析

目的 利用基因芯片技术寻找哮喘病患者与正常人外周血单核细胞之间差异表达基因,拟为哮喘的早期诊断及预防提供分子标记.方法 用淋巴细胞分离液分别提取16例哮喘病患者与16例正常人外周血单核细胞,用QIAGEN Rneasy Kit提取纯化样本总RNA,并合成用荧光标记的cRNA,分别与含有41 000条基因序列的全基因芯片杂交,以基因表达倍数值≥2.0和基因表达倍数值≤-2.0为阈值来确定差异表达基因,然后用Genespring软件利用生物信息学方法对差异表达基因进行功能分类分析.结果 按P＜0.05差异显著性标准,从34 183条表达基因谱中,筛选出哮喘患者与正常对照差异表达2倍以上的基因有4177条,差异表达2倍以上已知与哮喘相关的基因有19条.经代谢途径分析发现这些差异基因主要涉及到炎症反应、免疫反应、防御反应、创伤反应、外部刺激反应等8大功能分类.结论 哮喘的发生涉及众多基因表达的改变,芯片技术可以有效地筛选出哮喘患者与正常对照的差异表达基因,对进一步探索哮喘的发病机制、有效的干预或逆转哮喘具有重要意义.     

MicroRNA and gene expression analysis of melatonin-exposed human breast cancer cell lines indicating involvement of the anticancer effect

MicroRNAs (miRNAs) are small, noncoding RNAs that play a crucial role in regulation of gene expression. Recent studies have shown that miRNAs implicated in initiation and progression of various human cancers, including breast cancer and also analysis of miRNA expression profiles in cancer provide new insights into potential mechanisms of carcinogenesis. Melatonin, N-acetyl-5-methoxytryptamine, is synthesized by the pineal gland in response to the dark/light cycle and has been known to act as a synchronizer of the biological clock. Melatonin has a variety of therapeutic effects, such as immunomodulatory actions, anti-inflammatory effects, and antioxidant actions. Furthermore, melatonin is reported to have an anticancer function including suppression of the metabolism of tumor cells and induction of tumor suppressor genes in cancer cells, including breast cancer cells. In this study, we determined whether miRNAs play a role in regulation of various gene expression responses to melatonin in MCF-7 human breast cancer cells. We examined whole-genome miRNA and mRNA expression and found that 22 miRNAs were differentially expressed in melatonin-treated MCF-7 cells. We further identified a number of mRNAs whose expression level shows a high inverse correlation with miRNA expression. The Gene Ontology (GO) enrichment analysis and pathways analysis were performed for identification of the signaling pathways and biological processes affected by differential expression of miRNA and miRNA-related genes. Our findings suggested that melatonin may modulate miRNA and gene expression as an anticancer mechanism in human breast cancer cells.     

Serial analysis of gene expression in a rat lung model of asthma

Background and objective: The pathogenesis and molecular mechanism underlying asthma remain undetermined. The purpose of this study was to identify genes and pathways involved in the early airway response (EAR) phase of asthma by using serial analysis of gene expression (SAGE). Methods: Two SAGE tag libraries of lung tissues derived from a rat model of asthma and controls were generated. Bioinformatic analyses were carried out using the Database for Annotation, Visualization and Integrated Discovery Functional Annotation Tool, Gene Ontology (GO) Tree Machine and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Results: A total of 26 552 SAGE tags of asthmatic rat lung were obtained, of which 12 221 were unique tags. Of the unique tags, 55.5% were matched with known genes. By comparison of the two libraries, 186 differentially expressed tags (P < 0.05) were identified, of which 103 were upregulated and 83 were downregulated. Using the bioinformatic tools these genes were classified into 23 functional groups, 15 KEGG pathways and 37 enriched GO categories. Conclusions: The bioinformatic analyses of gene distribution, enriched categories and the involvement of specific pathways in the SAGE libraries have provided information on regulatory networks of the EAR phase of asthma. Analyses of the regulated genes of interest may inform new hypotheses, increase our understanding of the disease and provide a foundation for future research.     

胶原诱导性关节炎大鼠滑膜基因表达谱的cDNA微阵列研究

目的研究胶原诱导性关节炎大鼠滑膜基因表达谱，探讨类风湿关节炎的发病机制。方法用含588个基因的大鼠基因表达谱芯片研究胶原诱导性关节炎及正常大鼠滑膜基因表达谱。结果胶原诱导性关节炎大鼠差异表达基因共有68个，上调基因有63个，下调基因有5个。结论胶原诱导性关节炎是一个涉及多基因表达异常的全身免疫性疾病，筛选到的差异表达基因将为进一步研究类风湿关节炎发病机制及防治提供重要线索。     

Requirement for the histone deacetylase Hdac3 for the inflammatory gene expression program in macrophages

Histone deacetylases (HDACs) regulate inflammatory gene expression, as indicated by the potent antiinflammatory activity of pan-HDAC inhibitors. However, the specific contribution of each of the 11 HDAC proteins to the inflammatory gene expression program is unknown. Using an integrated genomic approach, we found that Hdac3-deficient macrophages were unable to activate almost half of the inflammatory gene expression program when stimulated with LPS. A large part of the activation defect was attributable to loss of basal and LPS-inducible expression of IFN-β, which maintains Stat1 protein levels in unstimulated cells and acts in an autocrine/paracrine manner after stimulation to promote a secondary wave of Stat1-dependent gene expression. Loss of Hdac3-mediated repression of nuclear receptors led to hyperacetylation of thousands of genomic sites and associated gene derepression. The up-regulation of the constitutively expressed prostaglandin endoperoxide synthase, Ptgs1 (Cox-1), a nuclear receptor target, had a causative role in the phenotype because its chemical inhibition reverted, albeit partially, the Ifn-β activation defect. These data indicate a central role for Hdac3 in inflammation and may have relevance for the use of selective Hdac inhibitors as antiinflammatory agents.     

Statistical limitations to the Cornell model of latent tuberculosis infection for the study of relapse rates

The Cornell model has been extensively used as a mouse model for studying the latent stage of Mycobacterium tuberculosis infection. In this model mice are infected and then given a course of chemotherapy prior to reactivation of infection. We discuss here the importance of using adequate mouse numbers in a Cornell model for the study of relapse rates in order to obtain sufficient statistical power to confirm a hypothesis. Experiments with small sample sizes are useful for 'screening' experiments, but will have very little value in 'confirming' the objective. When the objective of the experiment is confirmation of an effect through establishment of statistical significance, power calculations are critical in order to assure that the sample size will be sufficient to meet that objective.     

Different agonists recruit different stromal interaction molecule proteins to support cytoplasmic Ca2+ oscillations and gene expression

Stimulation of cells with physiological concentrations of calcium-mobilizing agonists often results in the generation of repetitive cytoplasmic Ca(2+) oscillations. Although oscillations arise from regenerative Ca(2+) release, they are sustained by store-operated Ca(2+) entry through Ca(2+) release-activated Ca(2+) (CRAC) channels. Here, we show that following stimulation of cysteinyl leukotriene type I receptors in rat basophilic leukemia (RBL)-1 cells, large amplitude Ca(2+) oscillations, CRAC channel activity, and downstream Ca(2+)-dependent nuclear factor of activated T cells (NFAT)-driven gene expression are all exclusively maintained by the endoplasmic reticulum Ca(2+) sensor stromal interaction molecule (STIM) 1. However, stimulation of tyrosine kinase-coupled FCεRI receptors evoked Ca(2+) oscillations and NFAT-dependent gene expression through recruitment of both STIM2 and STIM1. We conclude that different agonists activate different STIM proteins to sustain Ca(2+) signals and downstream responses.     

Microarray-based gene expression profiling in pancreatic ductal carcinoma: status quo and perspectives

Background Pancreatic ductal adenocarcinoma has an extremely poor prognosis. To improve the prognosis, novel molecular markers and targets for earlier diagnosis and adjuvant and/or neoadjuvant treatment need to be identified. One of the key techniques that has been developed to achieve this goal is DNA microarray profiling, which is used to identify the mechanisms of deregulated molecular functions in pancreatic carcinoma cells.Objective As several studies using microarrays have already been published, this review attempts to compare published data and crossvalidate the results. In addition, the applied techniques are discussed.     

Effects of TGFbeta1 on gene expression in the HP75 human pituitary tumor cell line identified by gene expression profiling

The pathogenesis of pituitary adenomas and many of the genes influencing growth of these tumors are unknown. TGFβ is known to inhibit proliferation of cultured anterior pituitary cells and anterior pituitary tumors, but the signal transduction pathways involved in the inhibition of growth are unclear. We treated the human HP75 pituitary cell line with 10⁻⁹ M TGFβ1 for 4, 24, and 96 h and performed global gene expression profiling by Affymetrix GeneChip microarray analysis. Quantitative PCR validation of specific genes involved in the TGFβ1-induced regulation of pituitary cell growth was also done. Of the 15,000 genes queried, there were 37 genes up-regulated and 48 genes down-regulated twofold or more after 4 h of TGFβ1 treatment. There were 121 genes up-regulated and 109 genes down-regulated twofold or more after 24 h of TGFβ1 treatment and 112 genes up-regulated and 43 genes down-regulated twofold or more after 96 h of TGFβ1 treatment. Galectin-3 (Gal-3) protein was decreased by TGFβ1 treatment and several genes which interacted with Gal-3 including RUNX1 and WNT5B were up-regulated after TGFβ1 treatment. SOX4 was also up-regulated by TGFβ1 treatment. SMAD3, which is directly involved in the TGFβ signal transduction pathway, was down-regulated by TGFβ1 treatment. These findings highlight the diverse gene networks and pathways through which TGFβ operates in its effects on pituitary tumor cells.