Chen Guang-Zhang, Fan Fang-Fang, Deng Si-Qian, Xia Xin-Yi, Bian Xiao-Chuan, Ren Yi-Han, Wei Li
Anhui Key Laboratory of Infection and Immunity, Bengbu Medical College, Bengbu 233030, China.
School of Clinical Medicine, Bengbu Medical College, Bengbu 233030, China.
Sichuan Da Xue Xue Bao Yi Xue Ban. 2023 Jan;54(1):122-127. doi: 10.12182/20230160201.
To isolate extracellular vesicles (EVs) from ( ), to examine their morphology, particle size, and distribution, to study the effect of EVs derived from ( EVs) on intracellular reactive oxygen species (ROS) production and cytokine secretion in dendritic cells (DCs), and to make preliminary exploration of EVs' effect on the immune regulation of DCs.
EVs were obtained by ultrafiltration concentration and the protein concentration was determined by BCA assay. The morphology of EVs was observed through negative staining electron microscopy (EM). The particle size distribution and concentration of EVs were determined by nanoparticle tracking analysis (NTA). Mouse bone marrow was isolated through sterile procedures and mice myeloid DCs were induced and amplified by the combined use of recombinant mouse granulocyte-macrophage colony-stimulating factor (rm GM-CSF) and recombinant mouse interleukin-4 (rm IL-4). Then, morphological and immunophenotypic characterization was performed. After that, the DCs were treated with -EVs at different concentrations and CCK-8 assay was done to measure their effect on the survival rate of DCs and to identify the appropriate stimulation concentration for subsequent experimental procedures. The intracellular ROS levels of DCs were evaluated with DCFH-DA fluorescence probe and the cytokine secretion of DCs was determined by ELISA.
EM observation showed that -EVs isolated by ultrafiltration concentration were spherical vesicles of varied sizes, all being approximately 100 nm in diameter and displaying typical morphology. NTA results from NanoSight nanoparticle tracker showed that the peak particle size was 98.5 nm, that the average particle size was 110.2 nm, and that the particle size was mainly distributed between 68.4-155.7 nm. -EVs that were smaller than 250 nm accounted for 98.39% of the total. Mouse myeloid DCs directionally induced and amplified displayed typical DC phenotype and morphology, and the purity exceeded 85%. EM verified the abundance of microvilli and radial protuberance on the surface of DCs, which had uniform cytoplasm and clear nuclear membrane. Loaded with -EVs at different concentrations, including 10 , 10 , and 10 particles/cell, the DCs had significantly upregulated levels of intracellular ROS ( <0.05). In addition, -EVs induced the release of IL-1β and IL-6 in a dose-dependent manner ( <0.05).
We established in the study a technical process for the extraction of -EVs by ultrafiltration concentration and obtained -EVs with sound morphology, high purity, and concentrated particle size distribution. Furthermore, -EVs can upregulate the intracellular ROS level in DCs and induce the release of IL-1β and IL-6 in a dose-dependent manner.
从（ ）中分离细胞外囊泡（EVs），检测其形态、粒径及分布，研究源自（ EVs）的EVs对树突状细胞（DCs）内活性氧（ROS）产生及细胞因子分泌的影响，并初步探索EVs对DCs免疫调节的作用。
通过超滤浓缩获得EVs，采用BCA法测定蛋白浓度。通过负染电子显微镜（EM）观察EVs的形态。采用纳米颗粒跟踪分析（NTA）测定EVs的粒径分布和浓度。通过无菌操作分离小鼠骨髓，联合使用重组小鼠粒细胞-巨噬细胞集落刺激因子（rm GM-CSF）和重组小鼠白细胞介素-4（rm IL-4）诱导并扩增小鼠髓样DCs。然后，进行形态学和免疫表型鉴定。之后，用不同浓度的-EVs处理DCs，采用CCK-8法检测其对DCs存活率的影响，以确定后续实验合适的刺激浓度。用DCFH-DA荧光探针评估DCs内ROS水平，采用ELISA法测定DCs的细胞因子分泌。
EM观察显示，通过超滤浓缩分离的-EVs为大小不一的球形囊泡，直径均约为1００nm，呈现典型形态。NanoSight纳米颗粒跟踪仪的NTA结果显示，峰值粒径为98.5nm，平均粒径为110.2nm，粒径主要分布在68.4 - 155.7nm之间。小于250nm的-EVs占总数的98.39%。定向诱导并扩增的小鼠髓样DCs呈现典型的DC表型和形态，纯度超过85%。EM证实DCs表面有丰富的微绒毛和放射状突起，细胞质均匀，核膜清晰。用不同浓度的-EVs处理DCs，包括10 、10 和10 个颗粒/细胞，DCs内ROS水平显著上调（<0.05）。此外，-EVs以剂量依赖方式诱导IL-1β和IL-6的释放（<0.05）。
本研究建立了通过超滤浓缩提取-EVs的技术流程，获得了形态良好、纯度高且粒径分布集中的-EVs。此外，-EVs可上调DCs内ROS水平，并以剂量依赖方式诱导IL-1β和IL-6的释放。
