jejunal Peyer’s patch (JPP) gene expression relative to jejunal mesenteric lymph node (j-MLN) gene expression.

Although immune mechanisms have been described for Peyer’s patch function, in discriminating food nutrients and commensal microflora from pathogenic challenge, an understanding of gene expression patterns associated with normal intestinal homeostasis is lacking. The effects of commensal bacterial colonization on global gene expression in the small intestine of germ-free mice have been described (1, 2). However, no studies have compared expression patterns of various healthy lymphoid tissues in conventionally-raised pigs or other species. The objective of this study was to investigate the pattern of normal gene expression in jejunal Peyer’s patches (JPP) of healthy pigs. We hypothesized that gene expression of intact jejunal Peyer's patch is characterized by genes associated with absorption and secretory functions as well as genes associated with GALT-specific immune functions. Eight hybridizations were performed, with dye swaps of JPP mRNA from four juvenile pigs. Each hybridization compared JPP from an individual pig to the jejunal mesenteric lymph node reference sample, which was pooled from all juvenile pigs in the study. However, two of the hybridizations resulted in poor quality data, and were excluded from further analysis. Normalization, hierarchical clustering, data visualization, and statistical analysis were performed by GeneSpring version 6.2 (Silicon Genetics, Redwood, CA). GeneSpring standardized the intensities for each spot by subtracting the local background, and then normalized globally by locally weighted linear regression (Lowess). A Lowess curve was fit to the log-intensity versus log-ratio plot. 20.0% of the data was used to calculate the Lowess fit at each point. This curve was used to adjust the control value for each measurement. If the control channel was lower than 10 then 10 was used instead. Spots with a flag value of “0” were excluded. The intensity of negative control DNA-containing spots was used to additionally exclude low intensity data. Negative controls include the gene for Arabidopsis thaliana homeodomain-like protein (AY054571); 200 ng/microl Poly(dA) (Amersham, Piscataway, NJ); a 461 bp fragment of the kanamycin-resistant gene, amplified from the pET24b+ plasmid (Novagen, Madison, WI); PRRS virus strain VR-2332 (PRU87392) ORFs 2, 3, 4, 5, 6 and 7; pCMVSport 6 plasmid (Invitrogen, Carlsbad, CA); and pGEM-T plasmid (Promega, Madison WI). The mean intensity of the negative controls for both Cy3 and Cy5 was calculated for each slide, and spots with a mean intensity less than the mean intensity + 2SD were excluded from further analysis. After normalization, GeneSpring averaged replicate spots for each clone across hybridizations. GeneSpring used the Cross Gene Error Model, which was based on replicate values. A Student’s t-test with the Benjamini and Hochberg false discovery rate multiple testing correction (MTC) verified the difference between the natural log of the normalized gene expression ratio (JPP:j-MLN) and a ratio of 1.0. p-values less than 0.05 were considered significant. Series_references: 1.Fukushima K, Ogawa H, Takahashi K, Naito H, Funayama Y, Kitayama T, Yonezawa H, and Sasaki I. Non-pathogenic bacteria modulate colonic epithelial gene expression in germ-free mice. Scand J Gastroenterol 38: 626-634, 2003. 2. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, and Gordon JI. Molecular analysis of commensal host-microbial relationships in the intestine. Science 291: 881-884, 2001. Keywords: other

尽管已有研究阐明派尔集合淋巴结（Peyer’s patch）在区分食物营养物、共生菌群与致病性感染过程中的免疫机制，但目前仍缺乏对与正常肠道稳态相关的基因表达模式的认知。已有研究报道了共生菌群定植对无菌小鼠小肠全基因组基因表达的影响（1, 2）。然而，尚无研究对比常规饲养猪或其他物种中各类健康淋巴组织的基因表达模式。本研究旨在探究健康猪空肠派尔集合淋巴结（JPP）的正常基因表达模式。我们提出假说：完整空肠派尔集合淋巴结的基因表达特征，以与吸收、分泌功能相关的基因，以及与黏膜相关淋巴组织（GALT）特异性免疫功能相关的基因为主。本研究共完成8次芯片杂交，对4头幼猪的空肠派尔集合淋巴结mRNA进行荧光染料互换标记。每次杂交均将单头猪的空肠派尔集合淋巴结样本，与本研究中所有幼猪混合制备的空肠肠系膜淋巴结（j-MLN）参照样本进行对比。但其中2次杂交获得的数据质量不佳，被排除于后续分析之外。数据分析的标准化、层级聚类、数据可视化及统计分析均通过GeneSpring 6.2版本（Silicon Genetics公司，加州雷德伍德市）完成。GeneSpring通过减去局部背景值对每个探针点的信号强度进行标准化，随后通过局部加权线性回归（Lowess）进行全局归一化：将Lowess曲线拟合至对数强度-对数比值散点图，每个拟合点采用20.0%的数据进行计算，以此曲线调整每次检测的对照值。若对照通道的信号强度低于10，则以10替代该值。标记值为"0"的探针点将被排除。同时利用含阴性对照DNA的探针点的信号强度进一步剔除低强度数据。阴性对照包括：拟南芥（Arabidopsis thaliana）同源域样蛋白基因（AY054571）；200 ng/μL 聚脱氧腺苷酸（Poly(dA)，Amersham公司，新泽西州皮斯卡塔韦市）；从pET24b+质粒（Novagen公司，威斯康星州麦迪逊市）扩增得到的461 bp卡那霉素抗性基因片段；猪繁殖与呼吸综合征病毒（PRRS virus）VR-2332株（PRU87392）的开放阅读框2、3、4、5、6及7；pCMVSport 6质粒（Invitrogen公司，加州卡尔斯巴德市）；以及pGEM-T质粒（Promega公司，威斯康星州麦迪逊市）。每张芯片的Cy3与Cy5通道阴性对照平均信号强度均被计算，信号强度低于平均强度+2倍标准差的探针点将被排除于后续分析。归一化完成后，GeneSpring对每次杂交中每个克隆的重复探针点信号值进行平均。GeneSpring采用基于重复值的交叉基因误差模型。通过采用本杰米尼-霍赫伯格错误发现率（Benjamini and Hochberg false discovery rate）进行多重检验校正（MTC）的Student t检验，验证归一化后的基因表达比值（JPP:j-MLN）的自然对数与比值1.0之间是否存在显著差异。p值小于0.05被认为具有统计学显著性。系列参考文献：1. Fukushima K, Ogawa H, Takahashi K, Naito H, Funayama Y, Kitayama T, Yonezawa H, Sasaki I. 非致病性细菌可调控无菌小鼠的结肠上皮基因表达. 《斯堪的纳维亚胃肠病学杂志》38: 626-634, 2003. 2. Hooper LV, Wong MH, Thelin A, Hansson L, Falk PG, Gordon JI. 肠道内共生宿主-微生物关系的分子分析. 《科学》291: 881-884, 2001. 关键词：其他