胸腺瘤相关重症肌无力患者瘤旁胸腺上皮细胞外泌体对Treg细胞功能的影响

Effects of exosomes of paratumoral thymus epithelial cells on Treg cell function in patients with thymoma-associated myasthenia gravis

  • 摘要: 目的:探讨胸腺瘤相关重症肌无力(TAMG)患者瘤旁胸腺上皮细胞外泌体对调节 T 细胞(Treg)叉状头转录因子 P3(FOXP3)表达及分泌转化生长因子(TGF)-β1、白细胞介素(IL)-10功能的影响。方法:收集TAMG患者及先天性心脏病无胸腺疾病的患者(对照组)胸腺组织,分离培养胸腺上皮细胞,使用广谱角蛋白(p-CK)和波形蛋白(Vimentin)染色行免疫荧光鉴定;膜亲和柱法提取胸腺上皮细胞外泌体并行透射电子显微镜(TEM)、粒径分析(NTA)及western blotting检测鉴定;磁珠分选法分离胸腺上皮细胞中Treg细胞,流式细胞术检测纯度;Treg细胞与外泌体共培养,行外泌体染色示踪实验,检测FOXP3表达及培养上清液TGF-β1、IL-10水平。结果:分离细胞呈长梭形,生长良好,免疫荧光p-CK呈阳性,Vimentin呈阴性,细胞为胸腺上皮细胞。TEM、NTA、western blotting结果提示提取物为外泌体。流式细胞术检测Treg细胞纯度>90%,纯度较好。外泌体染色示踪结果提示Treg细胞可吞噬胸腺上皮细胞外泌体,与TAMG组胸腺上皮细胞外泌体共培养后Treg细胞FOXP3表达减少(P<0.01),TGF-β1、IL-10分泌减少(P<0.01)。结论:Treg细胞可吞噬TAMG瘤旁胸腺上皮细胞外泌体,导致FOXP3表达减少、分泌TGF-β1、IL-10减少,免疫抑制功能减弱,其可能参与TAMG的发病过程。

     

    Abstract: Objective: To investigate the effects of exosomes derived from paratumoral thymic epithelial cells on the expression of forkhead box protein P3 (FOXP3) and the secretion of transforming growth factor-beta1 (TGF-β1) and interleukin (IL)-10 by regulatory T cells (Tregs) in patients with thymoma-associated myasthenia gravis (TAMG). Methods: Thymic epithelial cells were isolated and cultured from TAMG patients and patients with congenital heart disease without thymus disease (control group), and identified by immunofluorescence staining with pan-cytokeratin (p-CK) and Vimentin. The exosomes were extracted from thymic epithelial cells by membrane affinity column and identified by transmission electron microscopy (TEM), nanoparticle tracking analysis (NTA) and western blotting. Tregs were isolated from thymic epithelial cells by magnetic bead sorting and their purity was detected by flow cytometry. Tregs were co-cultured with exosomes, and exosome staining and tracing experiments were performed to detect the expression of FOXP3 and the levels of TGF-β1 and IL-10 in the culture supernatant. Results: The isolated cells were spindle-shaped and grew well. Immunofluorescence showed that the cells were positive for p-CK and negative for Vimentin, and the cells were thymic epitheliallls. TEM, NTA and western blotting results suggested that the extracts were exosomes. The purity of Tregs detected by flow cytometry was more than 90%, with good purity. The results of exosome staining showed that Tregs could phagocytose exosomes from thymic epithelial cells. After co-culture with exosomes of thymus epithelial cells in TAMG group, the expression of FOXP3 in Tregs was decreased (P<0.01) and the secretion of TGF-β1 and IL-10 was decreased (P<0.01). Conclusion: Tregs can phagocytose exosomes of paratumoral thymic epithelial cells of TAMG, leading to decreased FOXP3 expression, decreased secretion of TGF-β1 and IL-10, and weakened immunosuppressive function, which may be involved in the pathogenesis of TAMG.

     

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