Fan Ruiling, Wu Kaiwen, Yang Jingwei, Zhu Binjun, Jiang Tao, Liu Yu, Yuan Bin, Liu Lüye, Sun Yueshan, Sun Xiaobin, Liu Liu, Luo Wencai, Zhou Chunyang, Guo Yuanbiao, Liu Lei
Institute of Materia Medica, School of Pharmacy, North Sichuan Medical College, Nanchong, Sichuan, 637007, People's Republic of China.
Department of Ultrasound, Shanghai Pulmonary Hospital, Tongji University School of Medicine, Shanghai, 200433, People's Republic of China.
Int J Nanomedicine. 2025 May 29;20:6929-6948. doi: 10.2147/IJN.S516605. eCollection 2025.
Small extracellular vesicles (sEVs) derived from gastric juice (GJ) have emerged as potential biomarkers for gastric disease. However, methods for isolating sEVs from GJ (gsEVs) remain underexplored.
This study employed four methods for isolating gsEVs: ultracentrifugation (UC), PEG6000 precipitation combined with UC (PEG-UC), UC combined with size exclusion chromatography (UC-SEC), and ultrafiltration combined with SEC (UF-SEC). The yield and purity of gsEVs isolated by each method were evaluated, and the proteomic profile of gsEVs was examined through label-free quantitative proteomics. Additionally, a series of validation experiments were conducted to identify specific biomarkers for gsEVs.
The results revealed that gsEVs isolated using the UC method exhibited the highest purity and the largest number of identified proteins compared to the other methods. Notably, the gastric tissue-specific peptide trefoil factor 2 (TFF2) was highly expressed in gsEVs isolated by all methods, suggesting that TFF2 may serve as a specific biomarker for gsEVs. Validation experiments showed that TFF2 was exclusively present in gsEVs and not in the GJ supernatant after UC. Furthermore, TFF2 was selectively expressed in gsEVs, but not in sEVs derived from other biofluids such as intestinal juice and plasma. The efficiency of TFF2 as a gsEVs biomarker was higher than the commonly used biomarker CD9, CD81, and Syntenin-1. Multi-omics analysis indicated that the functions of gsEVs carrying TFF2 were primarily associated with inflammation and cancer.
The UC method is suitable for isolating gsEVs, particularly for mass spectrometry-based proteomic analysis. The small peptide TFF2 may serve as a potential specific biomarker for gsEVs. This study offers new insights for research on sEVs in gastric diseases.
源自胃液（GJ）的小细胞外囊泡（sEVs）已成为胃病的潜在生物标志物。然而，从GJ中分离sEVs（gsEVs）的方法仍未得到充分探索。
本研究采用四种方法分离gsEVs：超速离心（UC）、聚乙二醇6000沉淀结合UC（PEG-UC）、UC结合尺寸排阻色谱（UC-SEC）以及超滤结合SEC（UF-SEC）。评估了每种方法分离的gsEVs的产量和纯度，并通过无标记定量蛋白质组学检测了gsEVs的蛋白质组图谱。此外，还进行了一系列验证实验以鉴定gsEVs的特异性生物标志物。
结果显示，与其他方法相比，使用UC方法分离的gsEVs纯度最高，鉴定出的蛋白质数量最多。值得注意的是，胃组织特异性肽三叶因子2（TFF2）在所有方法分离的gsEVs中均高表达，这表明TFF2可能作为gsEVs的特异性生物标志物。验证实验表明，TFF2仅存在于gsEVs中，UC后胃液上清液中不存在。此外，TFF2在gsEVs中选择性表达，但在源自其他生物流体（如肠液和血浆）的sEVs中不表达。TFF2作为gsEVs生物标志物的效率高于常用生物标志物CD9、CD81和Syntenin-1。多组学分析表明，携带TFF2的gsEVs的功能主要与炎症和癌症相关。
UC方法适用于分离gsEVs，特别是用于基于质谱的蛋白质组学分析。小肽TFF2可能作为gsEVs的潜在特异性生物标志物。本研究为胃病中sEVs的研究提供了新的见解。
