第一部分
多组学联合解析脂代谢为ITP患者糖皮质激素应答的相关因素及PPAR通路调控恢复激素敏感性的机制探索

摘要

背景：原发免疫性血小板减少症（immune thrombocytopenia, ITP）是一种因自身免疫系统紊乱，攻击自身血小板及巨核细胞导致的出血性疾病。糖皮质激素作为一线治疗药物，虽能通过广泛免疫抑制快速提升血小板计数，但部分患者存在反应低下或无反应，最终需要接受二线治疗。CD4⁺ T细胞作为介导炎症、调节免疫耐受的关键免疫细胞群，是ITP治疗的新兴靶点，但是对糖皮质激素应答的相关因素及机制尚不明确，具体分子特征及调控网络仍需深入解析。

目的：本研究旨在整合多组学技术，系统分析初诊ITP患者（尤其糖皮质激素抵抗者）外周血CD4⁺ T细胞的分子图谱，揭示其免疫微环境失调与激素治疗低反应性的潜在关联，为逆转耐药提供新靶点。

方法：（1）纳入初治ITP患者，抽取外周血样本，检测外周血免疫细胞群比例，分离血浆，通过磁珠分选获得纯度＞90%的CD4⁺ T细胞，之后给予糖皮质激素治疗并前瞻性随访疗效，根据治疗后2周的血小板水平将患者分为糖皮质激素敏感组与抵抗组，并继续随访6个月排除激素依赖患者。筛选出符合条件的患者后建立高通量多组学测序队列，进行血浆非靶代谢组学、血浆蛋白组学、CD4⁺T细胞蛋白组学和CD4⁺T细胞转录组学同批次测序。（2）基于R语言进行数据分析，包括PCA降维、归一化、聚类分析、差异表达基因/蛋白/代谢物分析（DESeq2/limma）、相关性分析、通路功能富集与注释（KEGG/GO/Reactome)、基因集评分等。（3）建立体外糖皮质激素刺激培养体系，根据多组学分析结果提供的线索，观察过氧化物酶体增殖物激活受体γ（peroxisome proliferators-activated receptor γ，PPARγ）激动剂罗格列酮联合地塞米松相比地塞米松单药对Jurkat细胞系和ITP患者来源CD4⁺ T细胞的影响。

结果：糖皮质激素抵抗组在治疗前表现出较高的低密度脂蛋白胆固醇、高密度脂蛋白胆固醇和总胆固醇水平，且与治疗2周后血小板呈负相关（p＜0.05）。测序队列共纳入糖皮质激素抵抗ITP患者4例，糖皮质激素敏感ITP患者8例，正常对照5例。血浆代谢组和蛋白组结果提示糖皮质激素抵抗组游离脂肪酸、载脂蛋白升高，甘油磷脂水平下降。细胞蛋白组分析显示糖皮质激素抵抗组CD4⁺ T细胞糖酵解功能明显活化，而线粒体脂肪酸β氧化酶CPT1A和PPAR通路显著下调，同时转录组结果表明该组CD4⁺ T细胞IL-6表达上调，PI3K-Akt-mTOR通路和IL-17通路激活。体外实验结果证明PPARγ激动剂罗格列酮能够增强地塞米松对ITP患者CD4⁺ T细胞增殖和分化的抑制作用。

结论：糖皮质激素抵抗ITP患者存在脂代谢紊乱，高脂环境中的CD4⁺ T细胞出现糖脂代谢重编程，表现为糖酵解升高，脂肪酸氧化下降，并伴有炎症信号通路活化。靶向激活PPARγ可以有效增强地塞米松体外免疫抑制效果。这项探索性研究的结果为糖皮质激素抵抗ITP患者的早期识别和后续逆转耐药提供了新策略。

第二部分
全国遗传性凝血因子Ⅴ缺乏症回顾性研究

摘要

背景：遗传性凝血因子Ⅴ（factor Ⅴ, FⅤ）缺乏症是一种由F5基因变异引起的常染色体隐性遗传的罕见出血性疾病，表现为FⅤ活性降低并伴随出血症状，目前的替代治疗方法为新鲜冷冻血浆（fresh frozen plasma, FFP），辅以止血、改善贫血等支持治疗。

目的：调查并分析中国全国遗传性FⅤ缺乏症患者的人口学特征、临床表现、治疗方案及基因型特点。

方法：基于中国国家血友病登记系统（Chinese National Hemophilia Registry）的录入信息，我们对45例遗传性FⅤ缺乏症患者现状进行电子病历回顾性分析及电话随访患者。

结果：共纳入45例FⅤ活性（FⅤ:C）降低的患者，FⅤ:C从低于检测下限到最高48%不等，划分为轻度（FⅤ:C＞10%，n=6）、中度（FⅤ:C 1%-10%，n=31）及重度（FⅤ:C＜1%，n=8）三组.有39例患者提供了完整的临床病史，其中9例为无症状患者，6例FⅤ:C＜10%的患者发生Ⅲ级严重出血事件。最常见的出血部位是牙龈（38.5%）和皮肤出血（33.3%）。38.9%的育龄女性患者存在月经过多。20例患者进行了基因二代测序，共检测到33个位于F5基因的变异，其中12种致病性或疑似致病性变异和1种意义不明变异为首次报道的新发变异。结合临床表型分析结果显示，影响凝血因子V轻链结构域的变异（相较于重链结构域）与更低的FⅤ:C活性显著相关（p=0.002）。新鲜冰冻血浆为主要的替代治疗方式，有19例患者因出血接受按需治疗，另有2例无症状患者于手术前进行预防性输注。

结论：本研究对中国遗传性FⅤ缺乏症患者进行了全面描述，新发现的基因突变丰富了亚洲人群F5基因变异谱。

Part One

Multi-omics Reveals Lipid Metabolism-Associated Glucocorticoid Resistance and PPAR-Mediated Sensitivity Restoration in Immune Thrombocytopenia

Abstract

Introduction: Primary immune thrombocytopenia (ITP) is an autoimmune hemorrhagic disorder characterized by immune-mediated destruction of platelets and megakaryocytes. Although glucocorticoids are the standard first-line treatment to rapidly increase platelet counts through broad immunosuppression, some patients exhibit a poor or no response and eventually require second-line therapies. CD4⁺ T lymphocytes, which play a central role in mediating inflammation and regulating immune tolerance, are emerging as potential targets for ITP treatment. However, the factors and mechanisms underlying glucocorticoid responses remain poorly understood, and the specific molecular characteristics and regulatory networks involved require further investigation.

Aim: The aim of this study was to integrate multi-omics to systematically analyze the molecular profile of CD4⁺ T cells from newly diagnosed/treated ITP patients, particularly those resistant to glucocorticoid therapy. By uncover the potential link between immune microenvironment dysregulation and resistance to glucocorticoid, we aim to identify novel targets for overcoming glucocorticoid resistance in ITP.

Methods: (1) Newly diagnosed/treated ITP patients were enrolled, and peripheral blood samples were collected. The proportion of immune cell populations in peripheral blood was assessed, and plasma was separated. CD4+ T cells with >90% purity were isolated using magnetic bead sorting. Glucocorticoid therapy was administered, and patients were prospectively followed and categorized into a glucocorticoid-sensitive group and a resistance group based on platelet levels at two weeks after treatment. A six-month follow-up was conducted to exclude glucocorticoid-dependent patients. A high-throughput multi-omics sequencing program was established to analyze (i) plasma untargeted metabolomics, (ii) plasma proteomics, (iii) CD4+ T cell proteomics, and (iv) CD4+ T cell transcriptomics in parallel. (2) Data analysis was performed using R programming, which included dimensionality reduction (PCA), normalization, clustering analysis, differential expression analysis for genes (DESeq2/limma), proteins, and metabolites, correlation analysis, pathway functional enrichment and annotation (KEGG/GO/Reactome), gene set scoring, and other statistical methods. (3) Guided by multi-omics findings, an in vitro glucocorticoid-stimulated culture system was developed to evaluate the effects of the PPARγ agonist rosiglitazone combined with dexamethasone, compared to dexamethasone monotherapy, on the Jurkat cell line and CD4+ T cells derived from ITP patients.

Results: The glucocorticoid-resistant group exhibited higher levels of LDL, HDL, and total cholesterol before treatment, which were negatively correlated with platelet counts at two weeks post-treatment (p<0.05). The multi-omics profiling cohort included 4 glucocorticoid-resistant ITP patients, 8 glucocorticoid-sensitive ITP patients, and 5 healthy controls. Plasma metabolomics and proteomics analyses revealed that the glucocorticoid-resistant group had elevated levels of free fatty acids and apolipoproteins, while glycerophospholipid levels were decreased. CD4⁺ T cell proteomics identified enhanced glycolytic activity and suppressed fatty acid β-oxidation (CPT1A) and PPAR signaling pathway in the glucocorticoid-resistant group. Transcriptomic profiling demonstrated IL-6 overexpression (logFC=3.49，p=0.022) and upregulated PI3K-Akt-mTOR and IL-17 signaling pathways in the glucocorticoid-resistant group. Functional validation through in vitro assays confirmed that the PPARγ agonist rosiglitazone enhanced the suppression of dexamethasone on the proliferation and differentiation of CD4⁺ T cells.

Conclusion: Patients with glucocorticoid-resistant ITP exhibit disturbances in lipid metabolism, with CD4+ T cells in high-lipid environments undergoing reprogramming of glycolipid metabolism. This reprogramming is marked by an increase in glycolysis, a decrease in fatty acid oxidation, and the activation of inflammatory signaling pathways. Targeted activation of PPARγ significantly enhances the immunosuppressive effects of dexamethasone in vitro. These findings provide a novel approach for the early detection and potential reversal of glucocorticoid resistance in ITP patients.

Part Two

A Nationwide Study on Factor V Deficiency in China: Clinical Characteristics, Genotype, and Treatment Approaches

Abstract

Introduction: Congenital factor Ⅴ (FⅤ) deficiency is a rare bleeding disorder caused by recessive variants in the F5 gene. It is characterized by reduced FV activity and associated bleeding symptoms. The current treatment involves fresh frozen plasma (FFP) as a replacement therapy, along with supportive treatments such as hemostasis and anemia improvement.

Aim: To investigate the situation and genotype of patients with congenital FⅤ deficiency throughout China.

Methods: Based on data from the Chinese National Hemophilia Registry, we analyzed the clinical and genetic characteristics of 45 congenital FⅤ deficiency patients.

Results: The study cohort comprised 45 patients with confirmed FⅤ:C reductions (range: undetectable to 48%), including 6 mild (FⅤ:C > 10%), 31 moderate (FⅤ:C 1% to 10%), and 8 severe (FⅤ:C < 1%) patients. Of the 39 patients with available medical history, 9 were asymptomatic. Major bleeding (Grade III) occurred in 6 patients with FⅤ:C <10%. The most common symptoms included gingival (38.5%) and cutaneous bleeding (33.3%). Menorrhagia was observed in 38.9% of female patients of reproductive age. Gene sequencing identified 33 distinct variants in 20 patients, including 12 novel pathogenic/likely pathogenic variants and 1 novel variant of uncertain significance. Variants affecting the light chain (vs. heavy chain) were associated with lower FⅤ:C (p=0.002). Fresh frozen plasma served as the primary on-demand therapy, administered to 19 patients for active bleeding management and to 2 asymptomatic patients as preoperative prophylaxis.

Conclusion: The present study presented an overall view of FⅤ deficiency in China and supplemented the F5 gene variant spectrum in Asians.