目的分析早产和足月儿母乳来源外泌体（breast milk extracellular vesicles，BM-EV）的蛋白质组学差异。
方法收集2019年在南京医科大学附属妇产医院出生的早产儿和足月儿母乳各3例，采用超速离心法提取外泌体。初步鉴定外泌体特征后，采用液相色谱-串联质谱法（liquid chromatography-tandem mass spectrometry，LC-MS/MS）定量分析，筛选出早产儿BM-EV中显著上调的差异蛋白质（差异倍数≥1.5且 P<0.05），并进行GO和KEGG功能预测及相关信号通路分析。采用Mann-Whitney U检验或Fisher精确概率法进行组间比较。以Pearson相关分析描述样品间蛋白质定量值的相关性。使用 t检验比较2组样本间蛋白质丰度的差异，并对结果进行多重校正。应用超几何分布检验，筛选出差异蛋白质中显著富集的GO和KEGG通路条目。
结果（1）早产和足月组初产母亲分别为3例和1例。足月儿和早产儿BM-EV存在标记性蛋白分子分化簇9、分化簇81和热休克蛋白70。（2）6个样本组间可比性较好，组内重复性较高，样品间蛋白定量值的相关性最高达0.99。早产和足月儿样本的变异系数分别为11.21%和19.72%，且早产样本的变异系数比较集中。（3）鉴定得到945种蛋白质。早产与足月儿BM-EV之间存在156种差异蛋白质，其中在早产儿BM-EV中有83种上调。早产儿BM-EV中丰度值前3位的蛋白质分别为补体C4a、脂肪酸合成酶和硬化蛋白结构域蛋白-1。（4）GO功能预测中富集度最高的生物过程或细胞成分主要集中在血红蛋白和糖原的合成，参与免疫突触的构成，Fc γ受体信号通路介导的吞噬作用等。KEGG信号通路相关性最高的通路为核糖体相关通路、补体和凝血级联、中性粒细胞胞外陷阱形成和Fc γ受体介导的吞噬作用等。
结论早产儿BM-EV中显著上调的差异蛋白质可能通过参与调节早产儿的免疫、胃肠道功能和能量代谢过程，从而发挥保护作用。
ObjectiveTo analyze the differential expression of breast milk-derived extracellular vesicles (BM-EV) from mothers of preterm and term infants .
MethodsBreast milk samples were collected from preterm and term delivery (three cases in each) at the Women's Hospital of Nanjing Medical University in 2019. BM-EV was extracted using ultracentrifugation. After preliminary identification of the characteristics of BM-EV, liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used for protein quantification. Significantly up-regulated differential proteins (fold change≥1.5 and P<0.05) in the preterm group were screened. GO and KEGG were performed to predict the differentially expressed proteins' functional annotation and determine associated signaling pathways. Mann-Whitney U test and Fisher's exact test were used for intergroup comparisons. Pearson's correlation test describes the correlation of protein quantification values between samples. The differences in protein abundance were compared between the two groups using a t-test, followed by multiple corrections. Additionally, significantly enriched GO terms and KEGG pathways of the differentially expressed proteins were screened based on the hypergeometric distribution.
Results(1) There were three primiparae in the preterm group and one in the term group. Marker proteins CD9, CD81, and HSP70 were enriched in the BM-EV of both groups. (2) Six samples were comparable between groups and showed high reproducibility within groups. The correlation of protein quantification values between samples was up to 0.99. Furthermore, the coefficient of variation was 11.21% for preterm samples and 19.72% for term, and the data values in the preterm group were relative. (3) A total of 945 proteins were identified, and 156 were differentially expressed between preterm and term BM-EV, with 83 significantly up-regulated in preterm BM-EV. In the up-regulated proteins, the top three high-abundance proteins were complemented C4a, fatty acid synthase, and sclerostin domain-containing protein-1. (4) The biological processes or cellular components with the highest enrichment in GO functional prediction were mainly involved in hemoglobin and glycogen biosynthesis, immunological synapse formation, and phagocytosis mediated by the Fc γ receptor signaling pathway. The most relevant KEGG pathways were ribosome-related, complement and coagulation cascades, neutrophil extracellular trap formation, and Fc γ receptor-mediated phagocytosis.
ConclusionThe significantly up-regulated differential proteins in BM-EV may play a protective role by regulating immunity, gastrointestinal function, and energy metabolism in preterm infants.
姜承耀,严湘芸,陈文娟,等. 早产儿和足月儿母亲母乳来源外泌体的蛋白质组学差异分析[J]. 中华围产医学杂志,2023,26(02)：113-120.
DOI:10.3760/cma.j.cn113903-20220401-00317版权归中华医学会所有。
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姜承耀：实施研究、分析及解释数据、论文撰写及修改、统计分析；严湘芸、陈文娟：数据分析及论文审阅，数据收集，论文修改；韩树萍：研究设计和研究指导、数据分析、论文审阅及修改
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