Microarray identification of functional modules involving Haptoglobin and/or Hemopexin. Mus musculus

Haptoglobin and Hemopexin are plasma acute phase proteins that bind with high affinity hemoglobin and heme, respectively. Several studies have demonstrated that they have a key role in the protection against oxidative stress and inflammation. However, little is known about the functional modules in which they are involved. To gain insights into this issue, we integrated bioinformatic and experimental approaches to identify genes coexpressed with Haptoglobin or Hemopexin and modulated in Haptoglobin and/or Hemopexin knock-out mice. These genes have a high probability to be functionally related to Haptoglobin and/or Hemopexin. Keywords: haptoglobin, hemopexin, microarray, bioinformatics Overall design: We performed a gene expression analysis of the livers of Hp- and/or Hx-null mice compared to wild-type controls. The mice used in the following experiments, i.e. wild-type (Hp+/Hx+/), Hp-null (Hp-/-Hx+/), Hx-null (Hp+/Hx-/-), and HpHx-null (Hp-/-Hx-/-), were littermates derived by breeding F1 double heterozygous Hp+/-Hx+/- mice in the mixed genetic background C57/BLJ6 X 129Sv. We have three experimental points: Hx-null mice, Hp-null mice and HpHx-null mice. At least 5 adult mice per genotype were perfused via aorta with PBS, sacrificed and their liver pooled. Thirty microgram of total RNA were subjected to direct labeling reaction by incorporation of cyanin 3 (Cy3) or cyanin 5 (Cy5) fluorescent dyes into the cDNA by priming with oligo(dT). Four replicates were set up for each experimental point. In order to exclude artifacts resulting from different dye usage, we employed the dye-swap approach.

触珠蛋白（Haptoglobin）与血液结合素（Hemopexin）均为血浆急性期蛋白，二者分别以高亲和力结合血红蛋白（hemoglobin）与血红素（heme）。多项研究证实，二者在抵御氧化应激与炎症反应中发挥关键作用，但目前对二者所参与的功能模块仍知之甚少。为探究该问题，本研究整合生物信息学与实验方法，筛选与触珠蛋白或血液结合素共表达，且在触珠蛋白和/或血液结合素敲除（knock-out）小鼠中存在表达调控差异的基因，此类基因极有可能与触珠蛋白和/或血液结合素存在功能关联。
关键词：触珠蛋白、血液结合素、基因芯片（microarray）、生物信息学
实验设计：本研究针对触珠蛋白和/或血液结合素基因敲除小鼠的肝脏组织开展基因表达分析，并以野生型小鼠作为对照。本实验所用小鼠均为在C57/BLJ6 × 129Sv混合遗传背景下，通过繁育F1代双杂合子Hp+/-Hx+/-小鼠获得的同窝仔鼠，包含四种基因型：野生型（Hp+/Hx+/）、触珠蛋白单基因敲除（Hp-/-Hx+/）、血液结合素单基因敲除（Hp+/Hx-/-）以及双基因敲除（Hp-/-Hx-/-）。本实验设置三个实验组：血液结合素基因敲除组、触珠蛋白基因敲除组与双基因敲除组。每种基因型至少选取5只成年小鼠，经主动脉灌注磷酸盐缓冲液（PBS）后处死，收集肝脏组织并混合。提取30 μg总RNA，以寡聚(dT)为引物，通过将花青苷3（Cy3）或花青苷5（Cy5）荧光染料掺入互补脱氧核糖核酸（cDNA），完成直接标记反应。每个实验组设置4次生物学重复。为排除因荧光染料使用差异导致的实验伪影，本研究采用染料互换（dye-swap）实验策略。