研究背景和目的：针对全球人口面临的老龄化问题，深入研究衰老机制有助于揭示衰老的主要生物学特征，为预防和治疗衰老相关疾病提供关键的科学指导。骨髓作为多种造血干细胞、祖细胞的形成和功能单位，可作为研究衰老产生的增龄性变化的重要切入点。本研究旨在建立一种基于RNA测序阐明衰老过程中小鼠骨髓组织参与主要功能活动和细胞过程的特征性变化的研究方案，发掘用于衰老预测和干预的代表性潜在靶点。中性粒细胞作为造血干细胞的终末分化细胞，随着其吞噬、杀菌以及参与肿瘤微环境形成等功能的揭示，围绕中性粒细胞开发疾病治疗靶点逐渐成为热点，但是针对人类中性粒细胞成熟发育以及细胞凋亡的分子机制和功能联系仍缺少足够有效的研究论证。因此，建立更多有效且可行的细胞和动物模型来阐明中性粒细胞发育和凋亡的分子调控机制，将有助于中性粒细胞发育功能障碍相关疾病的治疗手段的开发。造血细胞特异性蛋白1相关蛋白X1（Hematopoietic cell-specific protein 1 associated protein X 1，HAX1）由常染色体隐性遗传重症中性粒细胞减少症的主要致病基因编码，可作为深入探究中性粒细胞功能影响的关键靶点。构建Haxl髓系特异性敲除的C57B/6J小鼠以及HAXI敲低和过表达的HL-60稳转细胞用于探究HAX1对中性粒细胞发育和凋亡的调控机制。

材料与方法：提取来自2月龄、10月龄和18月龄的各三只C57BL/6J雄鼠双侧胫骨和股骨的骨髓细胞，经过红细胞裂解处理后提取RNA用于RNA测序分析。采用慢病毒感染的方式构建HAX1敲低和过表达的HL-60稳转细胞株。通过ATRA联合DMSO对HL-60进行诱导分化，结合形态学鉴定与流式分析统计CD11b⁺中性粒细胞的占比和凋亡情况。利用Cre/Loxp技术构建 Hax1 基因髓系特异性敲除的小鼠模型，通过逆转录-实时荧光定量PCR（Reverse transcription-quantitative real-time PCR ，RT-qPCR）和Western blot在mRNA和蛋白水平上验证HAX1的表达。借助血常规、流式细胞术分析以及体外细胞集落形成实验明确HAX1对小鼠成熟免疫细胞、造血髓系分化以及中性粒细胞发育的影响。通过Western blot以及电镜分析确定了HAX1对中性粒细胞细凋亡的影响。利用RNA测序和RT-qPCR筛选确定了在转录水平受到HAX1敲除影响显著表达变化的下游基因，明确了HAX1调控中性粒细胞成熟发育以及细胞凋亡的主要通路。

结果：基因表达量和Venn分析的结果显示2 ~ 10月龄基因的表达变化以下调为主，10 ~ 18月龄则以上调为主，在小鼠成熟发育的过程中基因表达经历了由下调为主向上调为主的变化。通过京都基因与基因组百科全书的功能和富集分析以及基因集富集分析（Gene set enrichment analysis, GSEA）关注到的不同年龄阶段小鼠骨髓组织在“免疫系统”“发育和再生”“运输和分解代谢”“细胞生长和死亡”等通路存在显著的表达差异，其中在炎症、细胞骨架和DNA修复通路表现出持续激活，与逐渐下降的造血功能和波动的免疫调节形成对比。通过富集通路筛选出的差异表达上调的基因Bmpr1a和Inhba之间、差异下调的Dntt和Ccnd1之间以及Col1a1、Col1a2、Fcgr1、Fyn、Lgmn、Ctsl、Ctsk、Ctss、Gnail、Myl4和Ccr5之间均存在相互作用, 参与HSCs稳态、细胞周期、DNA损伤修复、免疫调节、细胞凋亡等功能调控Hax1髓系缺陷抑制骨髓粒细胞-单核细胞祖细胞（Granulocyte-monocyte progenitors，GMPs）导致终末分化髓系细胞下降，并且抑制中性粒细胞成熟导致未成熟中性粒细胞积累。此外，HAX1的缺少破坏中性粒细胞线粒体结构并且促进B淋巴细胞瘤-2（B-cell lymphoma-2，BCL-2）以及 caspase 酶家族促凋亡蛋白水平上升。RNA测序和mRNA水平分析显示HAX1缺失导致促凋亡的BAK1和介导线粒体自噬的Atg9a以及调控中性粒细胞功能的Toll样蛋白受体2 （Toll like receptor 2，TLR2）在转录水平显著上调，而调控细胞成熟转录因子PU.1显著下调。

结论：由于机体应对不断变化的体内外环境时复杂的调控机制，在衰老过程中小鼠骨髓组织在炎症反应、细胞骨架连接以及DNA损伤修复方面功能活性持续提升，而在造血功能维持方面调控不断下调，在免疫调节功能方面则表现波动性变化。小鼠骨髓免疫功能的减退与造血干细胞和祖细胞增殖和分化功能的下降密切相关，衰老后期免疫系统相关信号通路的表达上调更多源于机体细胞异常增殖导致肿瘤发生发展以及遭受病毒、细菌感染等其他多种恶性疾病概率增加。Bmpr1a、Inhba、Dntt、Ccnd1等多种差异表达的基因在HSCs的稳态和功能维持、细胞周期调控、DNA损伤修复、胞内信号传导、抗原的加工和呈递、细胞凋亡以及溶酶体和吞噬体的降解等功能方面存在精细网络调控，在保持特定功能独立性的同时，又在一定程度上表现出互补和协同效应。这些基因可以作为潜在的衰老标志物用于评估个体的衰老状态，预测衰老相关疾病的风险。HAX1通过BCL-2家族参与的线粒体介导凋亡通路调控中性粒细胞凋亡并且通过TLR2介导的PU.1水平调节影响中性粒细胞的发育。

Background and aims: In response to the global challenge of population aging, in-depth research into the mechanisms of aging can help uncover the primary biological characteristics of aging, providing crucial scientific guidance for the prevention and treatment of age-related diseases. The bone marrow, as the formation and functional unit of various hematopoietic stem cells and progenitor cells, serves as a significant point for studying the age-related changes with aging. The aim of this study is to establish a research protocol clarifying the characteristic changes in major functions and cellular processes involved in bone marrow during aging through RNA sequencing analysis, and to identify potential targets for aging prediction and intervention. Neutrophils, as terminally differentiated cells derived from hematopoietic stem cells, have gradually become a focal spot in developing treatment targets, as their functions such as phagocytosis, bactericidal activity, and tumor micro-environment formation continue to be uncovered. However, there still lacks sufficient and effective research on molecular mechanisms and functional connections related to the development and apoptosis of human neutrophils. Therefore, establishing more effective and feasible cellular and animal models to elucidate neutrophil maturation and apoptosis will aid in the treatments for diseases related to neutrophil developmental dysfunction. Hematopoietic cell-specific protein 1-associated protein X 1 (HAX1), encoded by the primary pathogenic gene of autosomal recessive severe congenital neutropenia, serves as a key target for in-depth exploration of neutrophil function. To investigate the molecular mechanisms that regulate neutrophil differentiation, development, and apoptosis, a conditionally Hax1 myeloid knockout C57BL/6J mouse model was constructed, along with stably-transduced HL-60 cell lines featuring HAX1 knockdown and over-expression.  

Methods: Bone marrow cells were extracted from the bilateral tibiae and femur of three C57BL/6J mice aged 2 months, 10 months, and 18 months. After red blood cell lysis, RNA was extracted for RNA sequencing analysis. The HL-60 cell lines with HAX1 knockdown and over-expression were constructed using lentiviral infection and induced to differentiate using ATRA and DMSO. The proportion and apoptosis status of neutrophils were assessed through morphological identification and flow cytometry analysis. The Hax1 gene conditionally knockout mouse model established by Cre/Loxp technology was proved at the mRNA and protein levels using reverse transcription-quantitative real-time PCR (RT-qPCR) and western blot. The impact of HAX1 on mature immune cells, myeloid differentiation and neutrophil development in mouse peripheral blood and bone marrow was elucidated through blood routine test, flow cytometry and colony forming assays. The role of HAX1 in neutrophil apoptosis was determined using western blot and electron microscopy analysis. RNA sequencing and RT-qPCR were employed to screen and identify downstream genes with significantly altered expression levels due to Hax1 knockout, clarifying the pathways through which HAX1 regulated neutrophil maturation and apoptosis.

Results: The results of gene expression and Venn analysis showed that gene expression levels were predominantly down-regulated from 2–10 months, but mainly up-regulated from 10–18 months. Gene expression thus changed from mainly down-regulation to mainly up-regulation during maturation and development in mice. Kyoto Encyclopedia of Genes and Genomes and gene set enrichment analyses indicated that bone marrow tissues in mice at different ages showed significant expression differences in the “immune system”, “development and regeneration”, “transport and catabolism”, “cell growth and death”, and other pathways. Specifically, Inflammatory, cytoskeletal, and DNA repair pathways showed sustained activation, contrasting with progressive hematopoietic decline and fluctuating immune regulation. Enriched pathway screening revealed interactions among differentially expressed genes, such us upregulated genes Bmpr1a and Inhba, downregulated genes Dntt and Ccnd1, and downregulated genes Col1a1, Col1a2, Fcgr1, Fyn, Lgmn, Ctsl, Ctsk, Ctss, Gnail, Myl4, and Ccr5, involved in HSC homeostasis, cell cycle, DNA repair, immune regulation, and apoptosis. HAX1 myeloid deficiency inhibited the differentiation of granulocyte-monocyte precursors (GMPs) leading to a decline in terminal differentiation myeloid cells and suppressing neutrophil maturation resulting in an accumulation of immature neutrophils. In addition, HAX1 deletion disrupted mitochondrial structure in neutrophils and increased the protein levels of B-cell lymphoma 2 (BCL-2) family members and caspase enzymes. RNA sequencing and mRNA analysis showed that the loss of HAX1 led to a significant upregulation of apoptosis promoting BAK1, mitochondrial autophagy mediateing Atg9a and Toll-like receptor 2 which regulates neutrophil function at the transcriptional level, while PU.1 regulating cell maturation was significantly downregulated.

Conclusion: Due to the body's complex regulatory mechanisms in adapting to internal and external changes, aging in mice leads to sustained increases in bone marrow activity related to inflammation, cytoskeletal connections, and DNA damage repair. However, hematopoietic regulation declines alongside fluctuating immunomodulation. This reduction in bone marrow immune-regulation correlates with diminished proliferation and differentiation of hematopoietic stem and progenitor cells. In late-stage aging, upregulated immune signaling pathways often result from aberrant cell proliferation, increasing susceptibility to tumors, infections, and other malignancies. Differentially expressed genes (e.g., Bmpr1a, Inhba, Dntt and Ccnd1) regulate HSC homeostasis, cell cycle, DNA repair, signal transduction, antigen processing, apoptosis, and lysosomal degradation. They form a regulatory network that maintains functional specificity while showing complementary and synergistic effects. These genes can serve as potential markers of aging for assessing an individual's aging status and predicting the risk of age-related diseases. HAX1 modulated neutrophil apoptosis through the mitochondrial-mediated apoptotic pathway involving the BCL-2 family and caspase enzymes and affected neutrophil maturation by regulating PU.1 levels via TLR2.