Effect of ILB on Gene expression in SHSY5Y neuroblastoma cells

The experiments were carried out to further understand the mechanism of action of ILB, a low molecular weight dextran sulphate, in neuronal cell. The experiments were carried out with ILB concentrations that are relevant to the therapeutic concentrations of the drug. The results indicate that ILB regulates the expression of genes to enhance cell survival and differentiation is a dose dependent manner. Upstream regulator analysis indicates that the gene expression changes are due to enhanced activity of growth factors and cytokines. Human SHSY5Y neuroblastoma cells (CRL-2266, ATCC) were cultured in DMEM:F12 medium supplemented with 10% FCS and 1% GlutaMAX™-I for 48 hours (day 0 timepoint). ILB was added to the cultures (final concentration: 0.01, 0.1, 1.0 and 10 µg/ml; n=3 for each concentration). Control cultures received only vehicle (0.9% NaCl in water added to culture medium 1:10,000; n=2). Cells were cultured for another 48 hours (day 2 timepoint). Samples were preserved in RNAlater. RNA was extracted using the Agilent RNA Isolation Kit. Expression analysis was performed using the SurePrint G3 Human Unrestricted GE 8x60K array (G4858A- 028004).

本实验旨在进一步解析低分子量硫酸葡聚糖ILB在神经元细胞中的作用机制。实验采用与该药物治疗浓度相符的ILB浓度开展。结果显示，ILB以剂量依赖方式调控基因表达，增强细胞存活与分化能力。上游调控因子分析表明，基因表达变化源于生长因子与细胞因子活性的增强。本实验使用人源SH-SY5Y神经母细胞瘤细胞（CRL-2266，ATCC），将其培养于添加10%胎牛血清（FCS）与1% GlutaMAX™-I的DMEM:F12培养基中，培养48小时后记为第0天时间点。随后向培养体系中加入ILB，设置其终浓度分别为0.01、0.1、1.0及10 µg/ml，每组设置3个生物学重复；对照组仅加入溶剂（按1:10000比例添加至培养基的0.9%无菌生理盐水，每组设置2个生物学重复）。继续培养48小时后，记为第2天时间点。所有样本保存于RNAlater试剂中。采用安捷伦RNA分离试剂盒（Agilent RNA Isolation Kit）提取总RNA。通过SurePrint G3人类全基因组表达8×60K微阵列芯片（G4858A-028004）完成基因表达分析。