Steroid Resistance in Nephrotic Syndrome

产品说明： 激素耐药型特发性肾病综合征（SRINS）是导致儿童肾功能衰竭和终末期肾病的重要原因。类固醇抵抗的原因尚不清楚，但有证据表明T细胞功能障碍是负责的。基于SRINS中异常T细胞NF-kB亚型组成的初步数据，拟议项目将确定导致SRINS患者糖皮质激素抵抗的T细胞信号传导机制缺陷。这些实验将比较新诊断的SRINS患者的T细胞与类固醇敏感患者和健康对照儿童的T细胞。将评价可能与异常NF-κ B功能相关的以下特异性信号转导机制的潜在缺陷：1）体外地塞米松暴露后糖皮质激素受体（GCR）的核转位; 2）STAT 5产生增加，并通过与STAT 5异源二聚化干扰GCR的核转移; 3）地塞米松刺激的IL-2产生; 4）响应于地塞米松暴露的凋亡诱导。这些方法将包括标准T细胞分离、用于基因表达的真实的时间RT-PCR、用于定量细胞核与细胞质组分中因子的电泳迁移率变动测定、用于检测复合GCR以及细胞凋亡的免疫沉淀测定

DESCRIPTION: Steroid-resistant idiopathic nephrotic syndrome (SRINS) is an important cause of renal failure and end-stage renal disease in children. The cause of steroid resistance is unknown but evidence suggests that T cell dysfunction is responsible. Based on preliminary data on abnormal T cell NF-kB isoform composition in SRINS, the proposed project will identify the defects in T cell signaling mechanisms that lead to glucocorticoid resistance in patients with SRINS. The experiments will compare T cells from newly diagnosed SRINS patients with those from steroid-sensitive patients and healthy control children. Potential defects in the following specific signal transduction mechanisms, which may be associated with abnormal NF-kB function, will be evaluated: 1) Nuclear translocation of the glucocorticoid receptor (GCR) in response to dexamethasone exposure in vitro; 2) Increased production of STAT5 and interference of nuclear transfer of GCR by heterodimerizing with STAT5; 3) Dexamethasone-stimulated IL-2 production; 4) Induction of apoptosis in response to dexamethasone exposure. The methods will include standard T cell isolation, real time RT-PCR for gene expression, electrophoretic mobility shift assay for quantifying factors in nuclear vs cytoplasmic fractions, immunoprecipitation assay to detect complexed GCRs, as well as apoptosis