唐氏综合征胎儿羊水细胞的着丝粒蛋白质组分析

doi:10.3969/j.issn.1674-8115.2026.01.002

上海交通大学学报（医学版） ›› 2026, Vol. 46 ›› Issue (1): 15-24.doi: 10.3969/j.issn.1674-8115.2026.01.002

唐氏综合征胎儿羊水细胞的着丝粒蛋白质组分析

周鹏¹^,², 刘艳娜¹, 颜景斌¹^,³()

^1.上海市儿童医院，上海交通大学医学院附属儿童医院，上海医学遗传研究所，上海 200040
^2.上海集爱遗传与不育诊疗中心，复旦大学附属妇产科医院，上海 200011
^3.国家卫生健康委员会医学胚胎分子生物学重点实验室，上海市胚胎与生殖工程重点实验室，上海 200040

收稿日期:2025-03-25 接受日期:2025-05-13 出版日期:2026-01-28 发布日期:2026-01-30
通讯作者: 颜景斌，研究员，博士；电子信箱：m18917128323@163.com。
作者简介:第一联系人：周鹏、刘艳娜、颜景斌参与实验设计，周鹏、颜景斌参与论文的写作和修改。所有作者均阅读并同意最终稿件的提交。
基金资助:
国家重大研发项目(2023YFC2705704);国家重大研发项目(2024YFC2707002);国家重大研发项目(2024YFC2707001);国家自然科学基金(81971421)

Centromere proteomic analysis of Down syndrome-derived amniocytes

Zhou Peng¹^,², Liu Yanna¹, Yan Jingbin¹^,³()

^1.Shanghai Institute of Medical Genetics, Shanghai Children's Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200040, China
^2.Shanghai Ji Ai Genetics and IVF Institute, Obstetrics and Gynecology Hospital, Fudan University, Shanghai 200011, China
^3.Key Laboratory of Embryo Molecular Biology, Ministry of Health of China; Shanghai Key Laboratory of Embryo and Reproduction Engineering, Shanghai 200040, China

Received:2025-03-25 Accepted:2025-05-13 Online:2026-01-28 Published:2026-01-30
Contact: Yan Jingbin, E-mail: m18917128323@163.com.
About author:First author contact:Zhou Peng, Liu Yanna, and Yan Jingbin contributed to the design of the experiments. Zhou Peng and Yan Jingbin were responsible for drafting the manuscript and conducting critical revisions. All authors have reviewed the final version of the manuscript and consented to its submission.
Supported by:
National Key Research and Development Program of China(2023YFC2705704);National Natural Science Foundation of China(81971421)

摘要/Abstract

摘要：

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。

关键词: 唐氏综合征, 羊水细胞, 着丝粒蛋白质组, 抗坏血酸过氧化物酶2邻近标记技术

Abstract:

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.

Key words: Down syndrome (DS), amniocyte, centromere proteome, APEX2 proximity labeling

中图分类号:

R394.3

周鹏, 刘艳娜, 颜景斌. 唐氏综合征胎儿羊水细胞的着丝粒蛋白质组分析[J]. 上海交通大学学报（医学版）, 2026, 46(1): 15-24.

Zhou Peng, Liu Yanna, Yan Jingbin. Centromere proteomic analysis of Down syndrome-derived amniocytes[J]. Journal of Shanghai Jiao Tong University (Medical Science), 2026, 46(1): 15-24.

图/表 4

图1 DS来源羊水细胞模型建立及CENPA的邻近标记蛋白的鉴定Note： A. Amniocytes culture (scale bar=25 µm). B. Transfection of amniocytes with the CENPA-EGFP-APEX2 plasmid (scale bar=100 µm). C. Colocalization of CENPA-EGFP-APEX2 with the centromeric marker protein CENPB (scale bar=5 µm). D. Western blotting analysis using HRP-conjugated streptavidin to detect biotinylated proteins in cell lysates from cells expressing CENPA-EGFP-APEX2. E. Proteins identified by LC-MS/MS in six samples. CON1‒CON3—three samples from the control group; DS1‒DS3—three samples from the DS group.

Fig 1 Establishment of DS-derived amniocytes models and identification of CENPA proximity-labeled proteins

表1 羊水细胞样本

Tab 1 Overview of amniocytes

Case	Maternal age/year	Gestation age/week	Fetal sex	Indication for testing	Chromosomal karyotype
1	40	16⁺³	Male	High-risk NIPT result	47, XY, +21
2	30	16⁺⁵	Male	High-risk NIPT result	47, XY, +21
3	44	18⁺²	Female	High-risk NIPT result	47, XX, +21
4	35	18⁺²	Male	High-risk NIPT result	47, XY, +21
5	41	19⁺³	Female	High-risk NIPT result	47, XX, +21
6	31	24⁺⁴	Female	High-risk NIPT result	47, XX, +21
7	32	13	Female	High-risk NIPT result	47, XX, +21
8	31	18⁺³	Male	High-risk NIPT result	47, XY, +21
9	25	16⁺¹	Male	High-risk NIPT result	47, XY, +21
10	38	18	Female	Advanced maternal age	47, XX, +21
11	46	24⁺⁵	Male	Positive screening for trisomy 21	46, XY
12	34	19⁺⁴	Male	Positive screening for trisomy 21	46, XY
13	33	21⁺⁶	Male	Positive screening for trisomy 21	46, XY
14	27	16⁺⁵	Male	High-risk serum markers	46, XY
15	30	17⁺⁵	Female	High-risk serum markers	46, XX
16	42	23⁺³	Male	Advanced maternal age	46, XY

图2 CENPA的邻近标记蛋白的功能注释Note： A. Protein functional annotation plot. B. KOG functional annotation plot. C. GO enrichment plot. D. KEGG pathway overview map. E. Protein domain annotation by InterProScan (IPR). F. Transcription factor annotation. CSD—cold-shock domain; zf-C2H2—zinc finger-C2H2; T-box—T-box domain; PC4—PC4 domain; HMG—high-mobility group; HMGI/HMGY—high-mobility group I/Y; MYB—myeloblastosis; STAT—signal transducer and activator of transcription.

Fig 2 Functional annotation of proximity-labeled proteins of CENPA

图3 DS组与正常组羊水细胞的CENPA邻近标记蛋白中的DEP富集分析Note： A/B. GO functional analysis of 32 DEPs. C. STRING protein-protein interaction network analysis of 32 DEPs. D/E. KEGG enrichment analysis of 32 DEPs. A/D. Up regulation. B/E. Down regulation. BP—biological process; CC—cellular component; MF—molecular function.

Fig 3 DEPs enrichment analysis of CENPA proximity-labeling proteins in amniocytes between the DS group and the normal group

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唐氏综合征胎儿羊水细胞的着丝粒蛋白质组分析

Centromere proteomic analysis of Down syndrome-derived amniocytes

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