RPS19异常相关DBA发病及地塞米松治疗DBA机制的研究

Diamond Blackfan anemia (DBA) is a congenital bone marrow failure syndrome characterized by erythroid hypoplasia due to impaired proliferation and differentiation of erythroid progenitors. Approximately 65% of DBA patients have heterozygous mutations or deletions in ribosomal protein (RP) genes with RPS19 being the first identified and the most commonly mutated gene. Due to difficulties in obtaining erythroid cells at sufficient quantity from DBA patients, erythroid cells from patients with DBA have not been well characterized and the mechanisms underlying the erythroid specific effects of RP associated DBA remain unclear. We have adopted ex vivo erythroid culture system from human CD34+ progenitor cells and developed methods to isolate erythroid cells at each distinct developmental stage from the ex vivo cultured erythroid cells. We also developed an inducible shRNA-mediated knockdown system. In this application, we will use these novel methods to establish RPS19-knockdown erythroid progenitor cellular models to examine the effect of RPS19 deficiency on erythroid progenitor function and to explore the underlying mechanisms. Furthermore, while it is well documented that dexamethasone has therapeutic effects on DBA, but mechanism of action of dexamethasone on DBA remains unclear. Therefore, we will also use RPS19-knockdown erythroid progenitor cellular models to define the cellular and molecular mechanisms for the therapeutic effects of dexamethasone on DBA. Successful accomplishment of our proposed studies will not only provide novel insights into mechanisms for the erythroid defects in DBA, but will also have significant clinic implications for the disease-specific treatments of DBA.

先天性纯红细胞再生障碍性贫血（DBA）是一种以单纯红系发育障碍为特征的骨髓衰竭综合症。研究发现DBA发病与核糖体蛋白异常导致红系祖细胞障碍相关，但由于未针对红系发育各特定阶段开展研究，对发病机制阐释尚不清楚。此外，尽管地塞米松（DX）已被用作DBA治疗首选药物，对DX作用机制认识仍不充分，精准治疗方案的制定缺乏依据。本课题组基于前期研究建立的造血干细胞向红细胞分化发育进程及阶段性调控机制的研究模型，拟通过在各阶段诱导性敲低RPS19表达，利用转录组深度测序结合多种功能验证实验深入探索RPS19突变影响红系发育的关键阶段及阶段特异性的调控机制；通过深入比对DX作用和未作用情况下RPS19敲低各阶段细胞增殖和分化等重要事件和转录谱的差异，阐明DX对RPS19诱发红系发育障碍的作用及其精细机制。本课题将为阐明RPS19突变引发红系发育障碍的精细机制以及探索DBA精准诊疗手段提供新视角和理论指导。

Diamond-Blackfan贫血（DBA）是一种核糖体蛋白功能障碍性疾病，其特征在于红系生成不良。25%患者表现为RPS19基因突变，但其在红系发育进程中作用机制还远不明晰。红系发育各阶段转录组数据揭示，RPS19在人红系发育不同阶段均有表达。目前的研究中，我们采用shRNA介导的方法来研究RPS19在人红细胞生成中的作用。结果显示，在人造血干细胞中敲低RPS19并诱导其向红系分化的进程中，细胞凋亡增加从而引起细胞扩增降低。我们进一步明确了RPS19敲低不仅影响红系早期祖细胞CFUE的增殖，而且对终末分化阶段原始红细胞的增殖也具有一定的影响。RPS19敲低影响红系祖细胞导致红系终末分化障碍。为了探究RPS19影响红系发育的机制，我们对正常组和RPS19敲低组进行RNA-seq。结果显示红系祖细胞中敲低RPS19造成细胞分化相关的转录因子GATA2的mRNA和蛋白水平升高，阻碍了GATA2对gata1基因表达的诱导，从而导致GATA1蛋白的表达的下降，进而导致红系祖细胞分化障碍。我们还发现RPS19敲低后与凋亡相关的基因如BAX、FAS mRNA表达上升，由此解释RPS19敲低后细胞凋亡的增加。为了探究其在小鼠体内红系发育进程中的作用，我们构建Gypa-eGFP-cre以及Gypa-tdTomato-cre小鼠模型，为RPS19在体内红系阶段性特异敲除提供有利工具。总而言之，我们的发现表明了RPS19在人类红细胞生成中的关键作用，并揭示了潜在的分子机制。此外，成功构建红系阶段特异性敲除工具鼠，为RPS19作用机制的进一步探索提供了重要的研究体系支撑。

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