Department of Biobank, Nanjing Drum Tower Hospital, The Affiliated Hospital of Nanjing University Medical School, Nanjing, People's Republic of China.
Department of Radiology, The Affiliated Cancer Hospital of Nanjing Medical University, Jiangsu Cancer Hospital, Jiangsu Institute of Cancer Research, Nanjing, People's Republic of China.
Biopreserv Biobank. 2024 Oct;22(5):506-516. doi: 10.1089/bio.2023.0070. Epub 2024 Mar 7.
Amniotic fluid (AF) plays a crucial role in diagnosing and predicting perinatal diseases, specifically preeclampsia (PE). Adequate preservation of AF samples is essential for advancing the development of PE-related biomarkers and understanding the disease's mechanisms. This study presents a method for preserving proteins in AF on a solid medium, specifically a nitrocellulose membrane, which is referred to as an AF membrane. Samples were collected from normotensive subjects and PE patients and treated with direct freezing and the AF membrane methods, respectively. Protein quality was assessed through sodium dodecyl sulfate-page and capillary electrophoresis. Liquid chromatography tandem mass spectrometry (LC-MS/MS) with data-independent acquisition was employed for proteomic analysis. Bioinformatics analysis identified differentially expressed proteins and pathways distinguishing normotensive subjects from PE patients. Comparison of the AF membrane method to the direct freezing method showed no significant impact on the protein content in the AF. The preservation methods employed did not result in evident protein differences or degradation in the AF obtained from both normotensive subjects and PE patients. Analysis based on Gene Ontology and HALLMARK gene sets revealed the upregulation of pathways associated with angiotensin, reactive oxygen species, and coagulation in PE patients. Furthermore, several biomarkers previously reported to be increased in PE serum, namely ENG, ERN1, FLT1, GDF15, HSPA5, LGALS3, PAPPA, PTX3, and SERPINE1, were significantly elevated in the AF. The AF membrane method proved to be highly effective, reliable, and durable for preserving proteins in AF samples. Preserving AF samples in a solid state holds significant value in discovering novel protein biomarkers and investigating the underlying mechanisms of PE.
羊水（AF）在诊断和预测围产期疾病，特别是子痫前期（PE）方面起着至关重要的作用。为了推进与 PE 相关生物标志物的开发和理解疾病的机制，充分保存 AF 样本至关重要。本研究提出了一种在固体介质（即硝酸纤维素膜）上保存 AF 中蛋白质的方法，称为 AF 膜。分别从正常血压受试者和 PE 患者中采集样本，并分别采用直接冷冻和 AF 膜方法进行处理。通过十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和毛细管电泳评估蛋白质质量。采用数据非依赖性采集的液相色谱串联质谱（LC-MS/MS）进行蛋白质组学分析。生物信息学分析鉴定了区分正常血压受试者和 PE 患者的差异表达蛋白和途径。将 AF 膜方法与直接冷冻方法进行比较，结果表明两种方法对 AF 中的蛋白质含量均无显著影响。这两种方法均未导致从正常血压受试者和 PE 患者获得的 AF 中出现明显的蛋白质差异或降解。基于基因本体论和 HALLMARK 基因集的分析表明，PE 患者中与血管紧张素、活性氧和凝血相关的途径上调。此外，一些先前报道在 PE 血清中升高的生物标志物，即 ENG、ERN1、FLT1、GDF15、HSPA5、LGALS3、PAPPA、PTX3 和 SERPINE1，在 AF 中显著升高。AF 膜方法在保存 AF 样本中的蛋白质方面非常有效、可靠且持久。将 AF 样本保存在固态中对于发现新型蛋白质生物标志物和研究 PE 的潜在机制具有重要价值。
