Li Xinqiang, Wang Xueteng, Cai Hailun, Wang Ye, Zhou Xin, Wu Bin, Cai Jinzhen, Teng Dahong
Organ Transplantation Center, Affiliated Hospital of Qingdao University, Qingdao 266100, Shandong, China.
Institute of Organ Donation and Transplantation, Medical College of Qingdao University, Qingdao 266100, Shandong, China.
Extracell Vesicles Circ Nucl Acids. 2025 Mar 7;6(1):141-157. doi: 10.20517/evcna.2024.52. eCollection 2025.
Exosomes derived from adipose-derived stem cells (ASCs) in mice have been reported to influence immune regulation. Yet, the potential immunological effects of ASCs-derived exosomes and their interaction with lymphocytes during transplant immunity remain understudied. ASCs from BALB/c mice, along with their conditioned culture medium, were collected for the extraction, isolation, and comprehensive characterization of exosomes. Splenic cell suspensions were isolated from BALB/c mice and subsequently processed for downstream analyses. Lymphocytes were isolated via gradient centrifugation and stimulated with the purified exosomes to assess their functional responses. Lymphocyte proliferation was quantified using the CCK8 assay, and the relative frequencies of CD4+ T cells, CD8+ T cells, Treg cells, NK (natural killer) cells, macrophages, B cells, dendritic cells (DCs), and Th17 cells were determined through flow cytometric analysis. Before establishing the skin transplantation model, the mice were administered PBS, 0.5 × 10 exosomes, 1 × 10 exosomes, 1.5 × 10 exosomes, or ASCs via intravenous injection through the tail vein. Seven days after transplantation, the spleens, drainage lymph nodes, and blood samples were harvested for lymphocyte isolation and further downstream analyses. Exosomes derived from ASCs significantly increased the CD4+/CD8+ ratio and Treg cell levels, without inducing any notable changes in Th17 cell content or CTLA-4 protein expression in CD4+ T cells. Compared to the PBS-treated group, both ASC and exosome treatment groups demonstrated an enhanced CD4+/CD8+ ratio, increased Treg cell content, and elevated CTLA-4 protein expression in spleen tissue following skin transplantation, while Th17 cell levels remained unaffected. Compared to the ASC treatment group, the exosome group exhibited a higher CD4+/CD8+ ratio and Treg cell levels, alongside a reduced proportion of PD-1+ Treg cells and lower CTLA-4 protein expression in CD3+CD4+ T cells. No significant differences were observed in the proportions of NK cells, macrophages, B cells, and DCs in the spleens across all treatment groups. In peripheral blood, an increased proportion of CD3+ T cells, macrophages, and DCs was detected, accompanied by a reduced proportion of NK cells and B cells. In the draining lymph nodes, no significant changes were observed in the proportions of CD3+ T cells and B cells, while macrophages, NK cells, and DCs showed elevated proportions. In the exosome-treated group, mouse grafts exhibited a disorganized and thinner granular layer, accompanied by focal regions of inflammatory cell infiltration. Both exosome and ASC treatments significantly extended the survival of skin grafts. Exosomes derived from ASCs promote lymphocyte proliferation and modulate their phenotypic profiles in mouse skin graft models, effectively extending graft survival.
据报道，小鼠脂肪来源干细胞（ASC）分泌的外泌体可影响免疫调节。然而，ASC来源的外泌体潜在的免疫效应及其在移植免疫过程中与淋巴细胞的相互作用仍未得到充分研究。收集BALB/c小鼠的ASC及其条件培养基，用于外泌体的提取、分离和全面表征。从BALB/c小鼠中分离脾细胞悬液，随后进行下游分析。通过梯度离心分离淋巴细胞，并用纯化的外泌体刺激以评估其功能反应。使用CCK8测定法对淋巴细胞增殖进行定量，并通过流式细胞术分析确定CD4+T细胞、CD8+T细胞、调节性T细胞（Treg）、自然杀伤（NK）细胞、巨噬细胞、B细胞、树突状细胞（DC）和Th17细胞的相对频率。在建立皮肤移植模型之前，通过尾静脉静脉注射给予小鼠PBS、0.5×10个外泌体、1×10个外泌体、1.5×10个外泌体或ASC。移植后7天，收获脾脏、引流淋巴结和血液样本用于淋巴细胞分离和进一步的下游分析。ASC来源的外泌体显著提高了CD4+/CD8+比值和Treg细胞水平，而未引起Th17细胞含量或CD4+T细胞中CTLA-4蛋白表达的任何显著变化。与PBS处理组相比，ASC和外泌体处理组在皮肤移植后脾脏组织中均表现出CD4+/CD8+比值升高、Treg细胞含量增加和CTLA-4蛋白表达升高，而Th17细胞水平未受影响。与ASC处理组相比，外泌体组表现出更高的CD4+/CD8+比值和Treg细胞水平，同时PD-1+Treg细胞比例降低，CD3+CD4+T细胞中CTLA-4蛋白表达降低。所有处理组脾脏中NK细胞、巨噬细胞、B细胞和DC的比例均未观察到显著差异。在外周血中，检测到CD3+T细胞、巨噬细胞和DC的比例增加，同时NK细胞和B细胞的比例降低。在引流淋巴结中，CD3+T细胞和B细胞的比例未观察到显著变化，而巨噬细胞、NK细胞和DC的比例升高。在外泌体处理组中，小鼠移植物表现出颗粒层紊乱且变薄，伴有炎症细胞浸润的局灶区域。外泌体和ASC处理均显著延长了皮肤移植物的存活时间。ASC来源的外泌体在小鼠皮肤移植模型中促进淋巴细胞增殖并调节其表型特征，有效延长移植物存活时间。
