Guangzhou University of Chinese Medicine, Guangzhou, China.
Department of Chinese Medicine, General Hospital of Southern Theater Command, Guangzhou, China.
Medicine (Baltimore). 2023 Aug 4;102(31):e34552. doi: 10.1097/MD.0000000000034552.
The comparative analysis of ultracentrifugation (UC) and polyethylene glycol (PEG)-based precipitation for the isolation of exosomes in gouty arthritis synovial fluid (GASF) is rarely reported, and it is not known whether different isolation methods can influence subsequent cytokine analysis.
GA patients were enrolled during a 1-year period from May 2021 to May 2022. Morphology, particle number, size, purity, protein concentration, and biomarker proteins of GASF-derived exosomes in both extraction methods were observed using transmission electron microscopy, nanoparticle tracer analysis, bicinchoninic acid assay, and Western blotting. An ELISA-based assay platform was used to detect the cytokines in exosomes using Meso Scale Discovery.
Thirty-two cases of fresh GASF were taken and randomly divided between the UC group (n = 16) and the PEG group (n = 16). Transmission electron microscopy images and nanoparticle tracer analysis results showed round vesicles measuring 100 nm on average. The protein expressions of TSG101, CD63, and CD81 in exosomes of the 2 groups were measured via Western blotting. The number and protein concentration of GASF-derived exosome particles from the PEG group were significantly higher than that of the UC group (P < .001). However, in the purity estimation, the UC group reflected significantly higher exosomes extractability (P < .01). Expression of IL-6 and IL-8 in the GASF-derived exosomes were higher in the UC group (P < .05), showing a median of 3.31 (interquartile range, IQR: 0.84-13.16) pg/mL, and a median of 2.87 (IQR: 0.56-13.17) pg/mL, respectively; moreover, IL-1β was mostly undetectable in the PEG group.
The UC method was found to yield exosomes of a higher purity, albeit at a lower quantity but with more abundant inflammatory cytokines; whereas the opposite was the case for the PEG group. The chemical precipitation method might not be suitable in terms of extracting GASF-derived exosomes for inflammation and immunity studies.
超离心（UC）和聚乙二醇（PEG）沉淀法在痛风性关节炎滑液（GASF）中外泌体分离的比较分析鲜有报道，且尚不清楚不同的分离方法是否会影响后续细胞因子分析。
2021 年 5 月至 2022 年 5 月期间，纳入了 32 例新鲜 GASF 患者，随机分为 UC 组（n=16）和 PEG 组（n=16）。采用透射电子显微镜、纳米颗粒示踪分析、二辛可宁酸法和 Western blot 观察两种提取方法所得 GASF 来源外泌体的形态、颗粒数、大小、纯度、蛋白浓度和生物标志物蛋白。采用 Meso Scale Discovery 基于 ELISA 的检测平台检测外泌体中的细胞因子。
透射电子显微镜图像和纳米颗粒示踪分析结果显示，平均直径为 100nm 的圆形囊泡。Western blot 法检测两组外泌体中 TSG101、CD63 和 CD81 的蛋白表达。PEG 组 GASF 来源外泌体颗粒数和蛋白浓度明显高于 UC 组（P<0.001）。然而，在纯度评估中，UC 组的外泌体提取率显著更高（P<0.01）。UC 组 GASF 来源外泌体中白细胞介素-6（IL-6）和白细胞介素-8（IL-8）的表达较高（P<0.05），中位数分别为 3.31（四分位距，IQR：0.84-13.16）pg/mL 和 2.87（IQR：0.56-13.17）pg/mL；此外，PEG 组中 IL-1β 大多无法检测到。
UC 法产生的外泌体纯度较高，但数量较少，但富含炎症细胞因子；而 PEG 组则相反。对于 GASF 来源的外泌体提取，化学沉淀法可能不适合用于炎症和免疫研究。
