Gene expression profiling of sciatic nerves from rats with diabetic peripheral neuropathy

To identify the key genes and pathways which might play critical roles in the pathogenesis of experimental DPN via microarray analysis. Adult male Sprague-Dawley rats (initial weight 250-300g, from the department of laboratory animal science of Fudan University) were randomly assigned into two groups: control rats and diabetic rats. Diabetes was induced with a single intraperitoneal injection of STZ (55mg/kg). Control rats were performed with a single intraperitoneal injection of 0.9% saline solution. Glucose level was evaluated using Sannuo strips on tail vein blood at day 3 after STZ injection and verified again at day 7 after STZ injection. Only rats with blood glucose level 16.7 mmol/L were considered diabetic.Six weeks after diabetes induction, nerve tissue samples (about 1cm long) were harvested from the right sciatic nerve of rats in control (n=3) and diabetic (n=3) groups for total RAN isolation. Microarray hybridization was then performed according to the Agilent One-Color Microarray-Based Gene Expression Analysis protocol (Agilent Technologies). Quantile normalization and subsequent data processing were performed using the GeneSpring GX v12.1 software package (Agilent Technologies). The univariate t-test with a fold-change >2 and P value < 0.001 was applied to identify the DEGs between control rats and diabetic rats. Credibility of the microarray data was validated through qRT-PCR on 4 genes. Total RNA was independently extracted from the right sciatic nerve of rats in both groups (n=3) 6 weeks after diabetes induction.本研究旨在通过微阵列分析（microarray analysis），筛选在实验性糖尿病周围神经病变（Diabetic Peripheral Neuropathy, DPN）发病机制中发挥关键作用的核心基因与信号通路。实验选取购自复旦大学实验动物科学部的成年雄性Sprague-Dawley（SD）大鼠，初始体重为250~300g，将其随机分为对照组与糖尿病模型组。糖尿病模型组大鼠通过单次腹腔注射链脲佐菌素（Streptozocin, STZ，55mg/kg）构建糖尿病模型，对照组大鼠则单次腹腔注射0.9%生理盐水。于STZ注射后第3天采集尾静脉血，采用三诺（Sannuo）血糖试纸检测血糖水平，并于第7天复测；仅血糖浓度达到16.7 mmol/L及以上的大鼠被认定为糖尿病造模成功。糖尿病造模6周后，分别从对照组（n=3）与模型组（n=3）大鼠的右侧坐骨神经中截取约1cm长的神经组织样本，用于总RNA（ribonucleic acid, RNA）提取。随后按照安捷伦（Agilent Technologies）单通道微阵列基因表达分析试剂盒说明书完成微阵列杂交。采用安捷伦GeneSpring GX v12.1软件包进行分位数归一化（quantile normalization）及后续数据分析。以倍数变化（fold-change, FC）>2且P值<0.001为筛选标准，通过单变量t检验鉴定对照组与糖尿病模型组大鼠的差异表达基因（Differentially Expressed Genes, DEGs）。为验证微阵列数据的可靠性，本研究于糖尿病造模6周后，再次独立从两组大鼠（每组n=3）的右侧坐骨神经中提取总RNA，并通过对4个基因开展实时定量逆转录聚合酶链反应（quantitative Real-Time Polymerase Chain Reaction, qRT-PCR）完成数据验证。