基于单细胞RNA-seq技术筛选肾小球系膜细胞增殖相关基因及临床应用研究

Mesangial proliferative glomerulonephritis (MesPGN) including IgA nephropathy (IgAN) is the most common primary glomerular disease with diverse clinical features and prognosis. Current standard transcriptome analysis approaches provide an average view of gene expression, and the genetic changes of specific cells are still unknown. The emerging single cell RNA-Seq technology profiles gene expression at the single cell level, and we suppose that it will serve as a powerful tool to screen differentially expressed genes of glomerular mesangial cells (GMCs) which are associated with the degree of mesangial proliferation and the clinical prognosis of MesPGN. Firstly, we will build MesPGN model using Meis1-EGFP mice that can be used to trace GMCs, obtain single GMCs using laser capture microdissection, and then identify differentially expressed genes between control and MesPGN model mice using single cell RNA-Seq and bioinformatics analysis. Secondly, we will uncover the function of target genes on GMCs proliferation and inflammatory factors secretion through over-expressing or low-expressing target genes in mouse mesangial cells. Finally, we will analyze the association between the expression of target genes in renal biopsy sections and clinical outcomes in IgAN cohort that is underway. The project is critical for identifying the new prognostic markers of MesPGN, and our results is promising in exploring novel pathomechanisms, drug discovery, and precisive therapy of MesPGN.

包括IgA肾病在内的系膜增殖性肾小球肾炎（MesPGN）是最常见的原发性肾小球疾病，临床表现多样，预后差异很大。目前常规基因组学方法研究MesPGN获得的是肾组织或肾小球不同类型细胞基因表达的平均值，难以明确特定细胞的基因表达。我们推测MesPGN系膜增殖程度和病情预后可能与不同程度病变部位肾小球系膜细胞（GMCs）的基因表达差异有关。本课题利用示踪GMCs的Meis1-EGFP小鼠建立MesPGN模型，激光显微切割获取单个GMCs，通过单细胞RNA-seq和生物信息学分析筛选系膜增殖相关差异表达基因；利用腺病毒转染和RNAi技术在小鼠系膜细胞内过表达和低表达靶基因，明确其对细胞增殖和分泌炎症因子的影响；利用已建立的IgA肾病队列明确靶基因表达与临床终点事件的关系。本研究有望获得评估MesPGN预后的新型标志物，对未来探索MesPGN病理机制、药物筛选和干预治疗具有重要的指导意义。

包括IgA肾病在内的系膜增殖性肾小球肾炎（MesPGN）是最常见的原发性肾小球疾病，临床表现多样，预后差异很大。目前常规基因组学方法研究MesPGN获得的是肾组织或肾小球不同类型细胞基因表达的平均值，难以明确特定细胞的基因表达。项目利用单细胞转录组测序技术首次绘制最常见的MesPGN ——IgA肾病肾脏单细胞基因表达谱。研究发现IgA肾病系膜细胞JCHAIN表达显著上调。已知J chain是IgA二聚化和跨上皮转运的关键分子，系膜细胞高表达J chain可能与骨髓来源的单体IgA1二聚化和系膜区特异性沉积有关。另外，研发发现提示系膜细胞高表达THY1、FN1等细胞外基质(extracellular matrix, ECM)分子和WFDC2分别与系膜细胞增殖、系膜基质增多和系膜基质清除下降有关。细胞配体-受体分析进一步提示IgA肾病系膜细胞与其他肾实质细胞、免疫细胞的细胞-细胞交流增加，这一现象反应了病变从系膜区向整个肾脏扩散的疾病进展过程。本研究可以帮助我们更加深入理解系膜增殖机制，而研究发现的差异表达基因可能作为潜在的干预靶点和预测预后的生物标志物。