基于单细胞转录组测序解析再生障碍性贫血造血干祖细胞的可变剪接异质性

Aplastic anemia is a bone marrow failure disease characterized by pancytopenia. Approximately 15% of aplastic anemia would further become myelodysplastic syndrome or acute myeloid leukemia. Recent studies have revealed high frequency mutations in splicing factors in hematopoietic stem and progenitor cells (HSPCs) of aplastic anemia, and our previous study showed that the important splicing factors in in HSPCs of aplastic anemia were significantly down-regulated. These evidences suggest that alternative splicing disorders are involved in the occurrence and development of aplastic anemia. However, the changes of alternative splicing in aplastic anemia and its underlying mechanisms remain unclear. This project will apply single-cell full-length transcriptome sequencing technology to perform alternative splicing studies on HSPCs of aplastic anemia. We plan to conduct further investigation with the following research goals: (1) to reveal a variable splicing regulatory network specific for different cell types of patient HSPCs; (2) to redefine HSPC cell types and discover new cell subtypes in aplastic anemia in the view of alternative splicing profiles and determine the cell type with the most significant splicing changes; (3) to exploit key splicing factors and alternative splicing targets specific for cell types of aplastic anemia; (4) to elucidate the molecular mechanism of alternative splicing in aplastic anemia. Overall, the alternative splicing research using single-cell full-length transcriptome sequencing will overcome the bottleneck of a small number of HSPCs of aplastic anemia, providing scientific basis for the pathological mechanism exploration and clinical diagnosis and treatment guidance for patients with aplastic anemia.

再生障碍性贫血（再障）是一种以全血细胞减少为特征的骨髓造血功能衰竭疾病。大约15%的再障会恶化成骨髓增生异常综合征或急性髓系白血病。近年来的研究表明再障造血干祖细胞中剪接因子高频突变，我们前期研究结果显示再障造血干祖细胞重要剪接因子显著下调表达。这提示我们可变剪接紊乱参与了再障的发生和发展。然而，可变剪接在再障造血干祖细胞中的变化及其作用机制仍不得而知。本项目将利用单细胞全长转录组测序技术，构建再障造血干祖细胞的可变剪接谱，拟开展以下新的研究：1）解析再障造血干祖细胞类型特异的剪接调控网络；2）重新定义再障骨髓中造血干祖细胞并鉴定新的细胞亚型,确定剪接变化最为显著的细胞类型；3）挖掘再障病变细胞类型特异的关键剪接因子及剪接靶基因；4）阐明可变剪接在再障中的致病分子机制。总之，单细胞分辨率的可变剪接研究将克服再障造血干祖细胞数量极少的瓶颈,为再障的病理机制探究和临床诊疗指导提供科学依据。

再生障碍性贫血（Aplastic Anemia, AA）（简称再障），是一组骨髓造血功能衰竭，导致外周血全血细胞减少的综合病征。随着病程进行，约15%的再障可进一步转化为恶性血液肿瘤如骨髓增生异常综合症（MDS）和急性髓系白血病（AML）。近年来的研究表明再障造血干祖细胞（HSPC）中剪接因子高频突变，再障造血干祖细胞重要剪接因子显著下调表达。这提示我们可变剪接紊乱参与了再障的发生和发展。由于再障患者骨髓残留HSPC数量极少，精细解析HSPC各组分在骨髓衰竭环境下病理性改变以及可变剪接作用机制存在较大挑战。. 我们首先从健康供者及再障患者骨髓及外周血中分选出CD34+造血干祖细胞，进行单细胞转录组测序（STRT-seq），随后根据转录组将HSPC定义为9类细胞亚群。基因表达和转录因子调控网络分析表明9类HSPC细胞亚群转录组的改变存在异质性，提示再障的谱系选择性造血抑制。此外，差异表达分析显示可变剪接信号在再障HSPC中发生了改变，而既往研究表明MDS也存在异常可变剪信号，且与MDS预后存在关联。为了研究再障的可变剪接及其与MDS之间的关系，研究人员又对再障患者骨髓HSPC细胞进行了单细胞全长转录组测序（Smart-seq2）。通过比较再障和MDS患者单细胞可变剪接事件，发现再障和MDS共同发生可变剪接改变的基因主要涉及DNA损伤修复。通过细胞表面受体和配体的基因表达，我们进一步探究了T淋巴细胞和HSPC的相互作用，发现再障中T淋巴细胞和HSPC相互作用显著增强。诱导凋亡的相互作用分子普遍存在重型再障骨髓细胞中。. 总之，我们从异质性角度解析了再障全谱系血细胞减少的分子基础，同时对患者的造血系统和免疫系统进行深入分析，提示异常可变剪接介导的DNA损伤修复信号的改变可能是再障恶性转化为MDS的关键因素，为再障患者免疫干预提供了新思路和潜在的新靶点。