STK10基因突变激活NF-κB/P53通路阻碍获得性PRCA红系分化的作用和机制研究

获得性纯红再障（PRCA）是一类红系前体细胞缺如的贫血性疾病，病程长易复发，但其发病机制不甚明确，基因突变是否参与发病亦无报道。我们预实验证实：全外显子测序在获得性PRCA患者中发现高频突变基因STK10，突变患者骨髓中STK10表达下调、P53上调；在K562细胞中慢病毒包装shRNA敲低STK10可影响其Hemin诱导的红系分化。因此，我们提出假设：获得性PRCA的基因突变损害造血干细胞向红系分化，在免疫损伤下最终发病。本项目拟：一、以shRNA敲低K562及CD34+细胞中STK10，CRISPR/Cas9对小鼠STK10点突变，并检测对红系、巨核系分化的作用；二、通过K562稳转株、PRCA患者样本、动物模型，探讨STK10突变对NF-κB/P53通路的调控机制。本研究旨在探索获得性PRCA的可能致病基因，及其在红系分化中的作用和机制，为临床精确诊断和探寻潜在治疗靶点提供理论依据。

Acquired pure red cell aplasia (PRCA) is anemia associated with the absence of erythroblasts and characterized by persistent and easily recurrence. However, the underline mechanisms of acquired PRCA remain obscure, and the role of gene mutation in the pathogenesis of acquired PRCA was not reported yet. Our previous studies confirmed that STK10 gene mutation is common in acquired PRCA patients, the mRNA/protein expression of STK10 reduced and P53 increased in the bone marrow of the patients which gene mutated. Furthermore, silence of STK10 gene through lentiviral vector harboring short hairpin RNAs in K562 cells could inhibit the erythropoiesis after induced by Hemin. Therefore, we make a hypothesis that the erythropoiesis of hematopoietic stem cells in acquired PRCA was impaired due to gene mutation, combined with the immunologic injury, ultimately leading to illness generation. In this proposed project we plan to 1. Silence the STK10 gene through lentiviral vector harboring short hairpin RNAs in K562 cells /primary cultured human CD34+ cells, and program a single-base substitution mutation of STK10 gene through CRISPR/Cas9 gene editing in C57BL/6 mice, then detect the erythropoiesis and megakaryocytopoiesis in these models, 2. Detect the regulation of NF-κB/P53 signal transduction due to STK10 mutated in these models and PRCA patient samples. In this research, we want to explore the possible driven gene mutation in acquired PRCA, and the role and mechanism of this mutation in the blockade of erythroid differentiation, which may provide evidence for precise diagnosis and exploring potential therapeutic targets in patients with acquired PRCA.