A novel approach to investigation of the pathogenesis of active minimal-change nephrotic syndrome using subtracted cDNA library screening
A novel approach to investigation of the pathogenesis of active minimal-change nephrotic syndrome using subtracted cDNA library screening
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DOI：
10.1681/asn.v1351238
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发表时间：
2002-05-01
影响因子：
13.6
通讯作者：
Guellaën, G
中科院分区：
文献类型：
--
作者：
Sahali, D;Pawlak, A;Guellaën, G
临床和实验观察表明，微小病变型肾病综合征（MCNS）是T细胞功能障碍的结果。通过未知的机制。用于鉴定可能参与MCNS的基因。从来自T细胞富集的外周血单核细胞的cDNA构建消减cDNA文库，所述T细胞富集的外周血单核细胞得自复发与缓解（“复发减去缓解”）期间的同一患者。该文库通过与正向（“复发减去缓解”）和反向（“缓解减去复发”）消减cDNA探针以及来自复发和缓解以及无关肾病综合征（膜性肾病）的未消减探针的差异杂交来筛选。共分离出84个转录本，其中12个未知功能的匹配蛋白和30个未知克隆。在已知的42份抄本中。至少18个与T细胞受体介导的复合物信号级联密切相关。包括编码T细胞受体组分和与细胞骨架支架相关的蛋白质的基因，以及转录因子。特别是，它被证明，Fyb/Slap，L-plastin，和grancalcin的表达水平在复发期间增加，这表明在T细胞接合后的近端信号的整合涉及这些细胞因子相关蛋白在MCNS中的优先募集。因为在复发样品中检测到非常低水平的白介素-12受体02 mRNA，所以白介素-12信号传导途径可能是有缺陷的，这表明在MCNS中，T细胞活化朝向T辅助细胞2表型演变。因此，差减克隆和差异筛选相结合的方法构成空气效率的鉴定基因，可能参与MCNS的病理生理过程。
Clinical and experimental observations suggest that minimal-change nephrotic syndrome (MCNS) results from T cell dysfunction. via unknown mechanisms. For the identification of genes that Lire potentially involved in MCNS. a subtractive cDNA library was constructed from cDNA from T cell-enriched peripheral blood mononuclear cells obtained from the same patient during relapse versus remission ("relapse minus remission"). This library was screened by differential hybridization with forward ("relapse minus remission") and reverse ("remission minus relapse") subtractive cDNAs probes, as well as unsubtracted probes from relapse and remission, and irrelevant nephrotic syndrome (membranous nephropathy). A total of 84 transcripts were isolated, of which 12 matched proteins of unknown function and 30 were unknown clones. Among the 42 known transcripts. at least 18 are closely involved in the T cell receptor-mediated complex signaling cascade. including genes encoding, components of the T cell receptor and proteins associated with the cytoskeletal scaffold, as well as transcription factors. In particular, it was demonstrated that the expression levels of Fyb/Slap, L-plastin, and grancalcin were increased during relapse, suggesting that the integration of proximal signaling after T cell engagement involves the preferential recruitment of these cytoskeleton-associated proteins in MCNS. Because very low levels of interleukin-12 receptor 02 mRNA were detected in relapse samples, the interleukin-12 signaling pathway might be defective, suggesting that, in MCNS, T cell activation evolves toward a T helper 2 phenotype. Therefore, the combination of subtractive cloning and differential screening constitutes air efficient approach to the identification of genes that are likely to be involved in the pathophysiologic processes of MCNS.