Transcription profiling of mouse thymus in pre diabetic and diabetic state of NOD (non obese diabetic) animal strain

The current project is within the range of molecular immunogenetic auto immune diseases and refers to the comparative study of promiscuous gene expression of tissue-specific antigens (TSAs) in the thymus of NOD mice line (non obese diabetic) who plays the auto-immune diabetes mellitus type 1, during the transition from state pre-diabetics to diabetics. Use the technology of oligo arrays to investigate the expression of miRNAs and cDNA microarrays to investigate the expression of genes encoding the messenger RNAs including TSAs (tissue specific antigens). Use the technology of oligo arrays to investigate the expression of miRNAs and cDNA microarrays to investigate the expression of genes encoding the messenger RNAs including TSAs (tissue specific antigens). Female NOD mice were born in specific pathogen free (SPF) conditions at the CEMIB-UNICAMP animal facility (University of Campinas, SP, Brazil) and maintained in SPF mini-isolators in our laboratory at the University of Sao Paulo, Campus of Ribeirao Preto, Brazil. We studied both pre-diabetic (8.2 week-old) and diabetic (20.2 week-old) animals. Diabetes was confirmed by blood glucose levels (?250 mg glucose/dL) using the Accu Check Active Kit (Roche Diagnostica Brasil, Sao Paulo, Brazil). The thymic stroma was separated from the whole thymus, as previously described (Gray et al. 2002). The central idea is to trace signatures of differential gene expression of the thymus at different stages (transition from state pre-diabetics to diabetics) and, using bioinformatics programs, applied to the analysis of data from arrays [Cluster & Tree View (for signatures of expression), SAM (for statistical analysis of the miRNAs and differentially expressed genes from TSAs), GenMiR++ and Cytoscape (to establish networks between miRNAs genes and genes of TSAs)].本项目隶属于分子免疫遗传学自身免疫病研究范畴，旨在针对非肥胖糖尿病（non obese diabetic, NOD）小鼠品系的胸腺组织中组织特异性抗原（tissue-specific antigens, TSAs）的异位基因表达（promiscuous gene expression）进行对比研究，该品系小鼠可自发1型自身免疫糖尿病，研究覆盖糖尿病前期至糖尿病发病的过渡阶段。本研究采用寡核苷酸芯片（oligo arrays）技术检测微小RNA（miRNAs）的表达水平，同时使用cDNA芯片技术分析编码包括组织特异性抗原在内的信使RNA（mRNA）的基因表达情况。 实验所用雌性NOD小鼠于巴西坎皮纳斯大学SPF级（specific pathogen free）动物实验中心（CEMIB-UNICAMP）繁育，随后在巴西圣保罗大学里贝朗普雷图分校实验室的SPF级独立通风笼具中饲养。本研究纳入糖尿病前期（8.2周龄）与糖尿病发病小鼠（20.2周龄），糖尿病状态通过罗氏活力型血糖检测试剂盒（Accu Check Active Kit，罗氏诊断巴西分公司，巴西圣保罗）检测血糖≥250 mg/dL予以确认。 按照Gray等人2002年发表的方法，从完整胸腺中分离胸腺基质。本研究的核心思路是解析小鼠胸腺在糖尿病前期至糖尿病发病的不同阶段的差异基因表达特征，并借助多款生物信息学工具对芯片数据进行分析：其中Cluster & Tree View用于可视化表达特征，SAM用于对微小RNA及组织特异性抗原相关差异表达基因进行统计学分析，GenMiR++与Cytoscape则用于构建微小RNA与组织特异性抗原编码基因之间的调控网络。