Transcription profiling of mouse thymus gland from pre-diabetic and diabetic animals

Female NOD mice were born in specific pathogen free (SPF) conditions at the CEMIB-UNICAMP animal facility (University of Campinas, Sao Paulo, 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 Diagnaœstica Brasil, Sao Paulo, Brazil). The thymic stroma was separated from the whole thymus. The mTEC 3.10 medullary thymic epithelial cell line was established from C57BL/6 mice, and the original medullary phenotype was confirmed by immunostaining with anti-cytokeratin monoclonal antibodies. In addition, the CD80+ phenotype was confirmed using fluorescent activated cell sorting (FACS) analysis. Cells were cultured in 10% fetal bovine serum-supplemented RPMI 1640 medium at 37oC and 5% CO2. Total RNA was extracted from 1x107 stromal cells (from pre-diabetic and diabetic animals) and 1x107 mTEC 3.10 cells using Trizol Reagent and following the manufacturer's instructions (Invitrogen, Carlsbad, CA, USA). RNA preparations were confirmed to be free of proteins and phenol using UV spectrophotometry and the state of degradation was assessed using agarose gel electrophoresis (ethidium bromide staining).雌性NOD小鼠（NOD mice）于巴西圣保罗大学坎皮纳斯分校CEMIB-UNICAMP动物设施的无特定病原体（specific pathogen free，SPF）环境中繁育，并饲养于巴西圣保罗大学里贝朗普雷图校区本实验室的SPF微型隔离器内。本研究纳入了糖尿病前期（8.2周龄）与糖尿病期（20.2周龄）两类实验动物。糖尿病状态通过血糖值＞250 mg葡萄糖/分升，结合罗氏诊断巴西分公司的Accu Check Active试剂盒进行确认。从完整胸腺中分离得到胸腺基质。mTEC 3.10髓系胸腺上皮细胞系由C57BL/6小鼠构建，其原始髓系表型经抗细胞角蛋白单克隆抗体免疫染色验证；此外，其CD80+表型通过荧光激活细胞分选（fluorescence activated cell sorting，FACS）分析得以确认。细胞于添加10%胎牛血清的RPMI 1640培养基中，在37℃、5%CO₂的培养条件下进行培养。分别从1×10⁷个糖尿病前期及糖尿病期小鼠来源的胸腺基质细胞，与1×10⁷个mTEC 3.10细胞中提取总RNA，操作严格遵循Trizol试剂（美国加利福尼亚州卡尔斯巴德市Invitrogen公司）的制造商说明书步骤。采用紫外分光光度法验证RNA制剂无蛋白质及苯酚污染，并通过琼脂糖凝胶电泳（溴化乙锭染色）评估RNA的降解状态。