7号染色体异常致造血细胞突变累积和免疫逃逸的机制研究

Cancer cells frequently exhibit chromosomal abnormalities, and specific cytogenetic aberrations often are predictors of outcome, as for example monosomy 7 in hematologic neoplasms. In bone marrow failure, chromosome 7 abnormalities (-7/7q-) are frequently associated with refractory cytopenias and progression to acute leukemia, and thus poor clinical outcome. According to previous findings, -7/7q- has been defined to comprise a distinct category of hematologic diseases. However, the functional consequences of aneuploidy at the cellular level are difficult to assess, due to lack of convenient markers to distinguish abnormal from diploid cells. We performed single-cell RNA sequencing (scRNA-seq) to study hematopoietic stem and progenitor cells (HSPCs) from patients with bone marrow failure and chromosome gain or loss. We distinguished -7/7q- cells from diploid cells in patient samples by computational analyses. We observed downregulation of genes involved in immune response, DNA damage checkpoint and apoptosis pathways, as well as increased SNPs in monosomy 7 cell, which may contribute to the clonal expansion of monosomy 7 cells with accumulated gene mutations. To further explore the role of -7/7q- in malignant transformation of hematopoietic cells, we designed experiments as following: establish -7/7q- NSG model using single-cell transplantation to separate patients’ -7/7q- cells and diploid cell; detect gene mutations, immune response and malignant transformation of -7/7q- cells in vivo and in vitro.

重现性染色体异常是肿瘤细胞的一个突出特征，常与疾病的预后相关。7号染色体异常(-7/7q-)是髓系恶性肿瘤最常见的染色体异常之一，患者多预后不良。在骨髓衰竭性疾病中，-7/7q-亦与难治性血细胞减少和疾病白血病进展有关。因此，伴有-7/7q-的血液病被归类于一个独特的群组。然而，由于缺乏特异性膜标志物，无法将异常细胞与正常细胞区分开来，这组疾病至今未能得到很好的理解。我们前期应用单细胞RNA测序检测骨髓衰竭伴染色体异常患者的造血干祖细胞，将-7/7q-细胞与正常核型细胞区分开，发现-7/7q-细胞的DNA损伤检查点、免疫应答和凋亡相关基因表达减低；增殖阶段细胞携带SNP数量显著增高。为进一步研究，我们计划通过NSG小鼠单细胞移植试验，构建伴-7/7q-的NSG小鼠模型，将患者-7/7q-细胞与正常核型细胞区分开来，定向检测-7/7q-细胞在体内及体外环境中的突变发生率、免疫应答及恶性转化。

重现性染色体异常是肿瘤细胞的一个突出特征，且常常与疾病的预后相关。在再生障碍性贫血（AA）等良性血液系统疾病中，染色体异常也可导致基因表达失常，从而促进疾病的发生、发展和恶性转化。AA被认为是细胞免疫介导的骨髓衰竭，目前AA病理机制推测为造血干细胞的抗原性发生改变，或外来抗原的交叉反应引起自身免疫T细胞克隆扩增，但AA存在异质性，引起异常免疫的抗原和相应淋巴亚群尚未明确。本项目通过对AA患者细胞遗传学特征分析和T细胞免疫组库特征分析，旨在探索AA克隆演进及免疫病理机制。结果发现染色体数量异常是AA患者诊断时发生染色体异常的主要类型。异常克隆持续存在的AA患者的免疫抑制治疗（IST）后6个月血液学反应低于染色体异常克隆消失的患者。伴有染色体异常克隆的AA患者IST后血液学反应、5年总生存、5年无事件生存、骨髓增生异常综合征或急性髓系白血病（MDS/AML）转化率与染色体正常患者无统计学差异。肝炎相关再生障碍性贫血（HAAA）患者和健康对照TRBV和TRBJ基因存在差异，高度扩增的T细胞克隆较健康对照增多，但HAAA并无较高频率表达的相同TCRβ CDR3氨基酸序列。HLA-DRB1*09:01可能是HAAA的风险等位基因。这些研究结果提示IST治疗后染色体异常克隆仍持续存在的AA患者的后续治疗应优先考虑HSCT而不是二次IST。HAAA可能不是由单一抗原刺激引起的疾病。此外，本项目针对AA患者免疫治疗和祛铁治疗机制做了初步的探讨探究。这些均为AA的基础理论和临床应用领域提供了新的思路。

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