目的·探索唐氏综合征（Down syndrome，DS）胎儿羊水细胞的着丝粒蛋白质组中的差异表达蛋白。方法·以DS胎儿及正常胎儿羊水细胞为模型，构建着丝粒蛋白A（centromere protein A，CENPA）-增强型绿色荧光蛋白（enhanced green fluorescent protein，EGFP）-抗坏血酸过氧化物酶2（ascorbate peroxidase 2，APEX2）融合蛋白慢病毒载体，转染羊水细胞，靶向标记着丝粒邻近蛋白；通过免疫荧光共定位判断目的蛋白表达位置，通过生物素化处理和蛋白质印迹法（Western blotting）验证蛋白标记情况。采用4D label-free定量蛋白质组学技术分析3例DS组与3例正常组样本，通过基因本体论（Gene Ontology，GO）、原核/真核同源蛋白簇（Clusters of Orthologous Groups/Eukaryotic Orthologous Groups，COG/KOG）及京都基因与基因组百科全书（Kyoto Encyclopedia of Genes and Genomes，KEGG）数据库对标记蛋白进行功能注释；以P<0.05且差异倍数（fold change，FC）对数的绝对值（|log₂FC|）>0.585筛选DS组和正常组羊水细胞差异表达蛋白，并通过STRING数据库构建蛋白质-蛋白质相互作用（protein-protein interaction，PPI）网络，鉴定核心枢纽蛋白。结果·成功建立DS羊水原代细胞着丝粒邻近蛋白标记模型，免疫荧光共定位和蛋白质印迹结果证明CENPA-EGFP-APEX2可精准锚定着丝粒并标记邻近蛋白。蛋白质组学共鉴定出999个高可信蛋白，生物信息学分析显示这些蛋白富集于氧化还原及癌症相关通路等。差异蛋白分析显示MX动力蛋白样GTP酶1（MX dynamin-like GTPase 1，MX1）、信号转导及转录激活蛋白1（signal transducer and activator of transcription 1，STAT1）等显著上调，β-肌动蛋白（actin β，ACTB）等下调；PPI分析揭示小泛素样修饰蛋白2（small ubiquitin like modifier 2，SUMO2）、核内不均一核糖核蛋白A/B（heterogeneous nuclear ribonucleoprotein A/B，HNRNPAB）和丝裂原活化蛋白激酶（mitogen-activated protein kinase，MAPK）为核心枢纽蛋白。结论·DS胎儿羊水细胞着丝粒蛋白质组中存在多种差异表达蛋白，其中MX1和STAT1异常激活、ACTB下调，这些差异蛋白之间的相互作用以SUMO2、MAPK、HNRNPAB为主要核心。

Objective ·To explore differentially expressed proteins in the centromere proteome of amniocytes from fetuses with Down syndrome (DS). Methods ·Amniocytes from DS and normal fetuses were used as models. A lentiviral vector encoding the centromere protein A-enhanced green fluorescent protein-ascorbate peroxidase 2 (CENPA-EGFP-APEX2) fusion protein was constructed and transfected into amniocytes to target and label centromere-proximal proteins. The expression and location of the target protein were determined by immunofluorescence co-localization, and protein labeling was verified by biotinylation treatment and Western blotting. Quantitative proteomics analysis using 4D label-free technology was used on three DS samples and three normal samples. Functional annotation of the labeled proteins was performed using the Gene Ontology (GO), Clusters of Orthologous Groups/Eukaryotic Orthologous Groups (COG/KOG), and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases. Differentially expressed proteins (DEPs) between DS and normal amniocytes were screened with thresholds of P<0.05 and |log₂FC|>0.585, and a protein-protein interaction (PPI) network was constructed using the STRING database to identify core hub proteins. Results ·A centromere-proximal protein labeling model using DS amniocytes was successfully established, and immunofluorescence co-localization and Western blotting confirmed that CENPA-EGFP-APEX2 was precisely anchored at the centromere and labeled proximal proteins. Proteomic analysis identified 999 high-confidence proteins, and bioinformatics analysis showed that these proteins were enriched in redox-related and cancer-related pathways. Differential expression analysis revealed significant upregulation of MX dynamin-like GTPase 1 (MX1) and signal transducer and activator of transcription 1 (STAT1), as well as downregulation of actin β (ACTB). The PPI network further identified small ubiquitin-like modifier 2 (SUMO2), heterogeneous nuclear ribonucleoprotein A/B (HNRNPAB), and mitogen-activated protein kinase (MAPK) as key hub proteins. Conclusion ·There are various differentially expressed proteins in the centromere proteome of DS fetal amniocytes, with abnormal activation of MX1 and STAT1, downregulation of ACTB, and interactions among these differential proteins centered on SUMO2, MAPK, and HNRNPAB.