探讨调节性T细胞(Treg)外泌体(rEXS)对内层血－视网膜屏障(IBRB)的保护作用。

BV2小胶质细胞未添加100 ng/ml脂多糖(LPS)和rEXS者设为对照组；分别将添加100 ng/ml LPS、10 μg/ml rEXS＋100 ng/ml LPS、20 μg/ml rEXS＋100 ng/ml LPS及50 μg/ml rEXS＋100 ng/ml LPS设为LPS刺激组、10 μg/ml外泌体组、20 μg/ml外泌体组及50 μg/ml外泌体组。构建BV2细胞和人脐静脉内皮细胞(HUVEC)的共培养系统模拟视网膜血管。设以未添加LPS和rEXS无BV2细胞的HUVEC新鲜培养基作为HUVEC对照组，以BV2＋HUVEC、100 ng/ml LPS ＋BV2＋HUVEC及50 μg/ml rEXS＋BV2＋HUVEC分别为共培养组、共培养刺激组及共培养外泌体组。采用酶联免疫吸附试验检测rEXS处理后白细胞介素(IL)－1β、肿瘤坏死因子(TNF)－α及IL－10的水平。采用免疫印迹法分析检测紧密连接蛋白(ZO)－1和闭合蛋白的表达水平。TNF－α、IL－1β、IL－10、ZO－1及闭合蛋白均符合正态分布，以±s表示，组间比较采用单因素方差分析及LSD事后检验。

分离rEXS的纳米示踪结果显示为钟形曲线，曲线下的大部分面积落在rEXS的特征性尺寸范围50～155 nm内，峰值在106 nm和155 nm处，与rEXS的粒径大小分布吻合。rEXS的浓度为4.74×10¹⁰个颗粒/ml。rEXS中存在特异性标记物。透射电子显微镜成像显示，颗粒的形状与rEXS一致且直径约100 nm。对照组、LPS刺激组、10 μg/ml外泌体组、20 μg/ml外泌体组及50 μg/ml外泌体组TNF－α蛋白浓度分别为(1035.96±32.36)pg/ml、(2965.25±4.08)pg/ml、(2960.41±35.65)pg/ml、(2863.32±30.55)pg/ml及(2586.32±33.25)pg/ml；IL－1β蛋白浓度分别为(2.60±0.05)pg/ml、(17.18±0.13)pg/ml、(10.98±0.06)pg/ml、(6.39±0.04)pg/ml及(4.05±0.06)pg/ml；IL－10蛋白浓度分别为(28.24±2.66)pg/ml、(24.55±0.77)pg/ml、(33.96±3.58)pg/ml、(34.29±4.32)pg/ml及(48.32±1.55)pg/ml，组间比较，差异有统计学意义(F＝23.01，1.96，29.53；P<0.05)。以HUVEC对照组为基数，共培养组、共培养刺激组及共培养外泌体组ZO－1蛋白相对表达量分别为1.28±0.010、0.8±0.02及1.23±0.01；闭合蛋白相对表达量分别为1.37±0.04、0.81±0.02及1.18±0.03，组间比较，差异有统计学意义(F＝808.3，222.2；P<0.05)。

rEXS能抑制TNF－α和L－1β释放，促进IL－10介导的抗炎效应，并缓解LPS诱导的紧密连接破坏，可能对IBRB有保护作用。

The aim of this study is to investigate the effect of extracellular vesicles isolated from T regulatory cells (rEXS) on the inner blood-retinal barrier (IBRB).

BV2 microglial cells were divided into five groups: control (no LPS or rEXS), LPS-stimulated (100 ng/ml LPS), and three rEXS-treated groups (10 μg/ml, 20 μg/ml, and 50 μg/ml rEXS+ 100 ng/ml LPS). A co-culture system of BV2 cells and human umbilical vein endothelial cell (HUVEC) was used to simulate retinal vessels, with groups divided as follows: HUVEC control (no BV2), co-culture (BV2+ HUVEC), co-culture stimulated (BV2+ HUVEC+ 100 ng/ml LPS), and co-culture exosome (BV2+ HUVEC+ 50 μg/ml rEXS). ELISA was used to measure interleukin (IL)-1β, tumor necrosis factor (TNF)-α, and IL-10 levels, while Western blot analyzed zona occludens (ZO)-1 and occludin expression. Data were expressed as ±s and compared by one-way ANOVA and LSD post-hoc test.

Nanoparticle tracking analysis showed that rEXS had a characteristic size range of 50 to 155 nm, with a peak at 106 nm and 155 nm. The rEXS concentration was 4.74×10¹⁰particles/ml. Specific markers were detected in rEXS. Transmission electron microscopy confirmed rEXS particles were approximately 100 nm in diameter. The TNF-αconcentrations in the control group, LPS-stimulated group, 10 μg/ml rEXS groups, 20 μg/ml rEXS groups, 50 μg/ml rEXS groups were (1035.96±32.36)pg/ml, (2965.25±4.08)pg/ml, (2960.41±35.65)pg/ml, (2863.32±30.55)pg/ml, (2586.32±33.25)pg/ml; IL-1βwere (2.60±0.05)pg/ml, (17.18±0.13)pg/ml, (10.98±0.06)pg/ml, (6.39±0.04)pg/ml, (4.05±0.06)pg/ml; IL-10 were (28.24±2.66)pg/ml, (24.55±0.77)pg/ml, (33.96±3.58)pg/ml, (34.29±4.32)pg/ml, and (48.32±1.55)pg/ml, respectively. There were significant differences between them (F=23.01, 1.96, 29.53; P<0.05). In the co-culture system, compared with relative control group, the ZO-1 expression levels in the co-culture group, co-culture stimulated group, co-culture exosome group were (1.28±0.010), (0.8±0.02), and (1.23±0.01); occludin were (1.37±0.04), (0.81±0.02), (1.18±0.03), respectively. There were significant differences between them (F=808.3, 222.2; P<0.05).

The results of the present study revealed that rEXS may have a protective effect on the IBRB by inhibiting the release of TNF-α and IL-1β cytokines, promoting IL-10-mediated anti-inflammatory effects, and alleviating LPS-induced tight junction disruption.