缺氧对肾小管上皮细胞分泌外泌体的影响

目的    观察缺氧对肾小管上皮细胞分泌外泌体的影响，探讨外泌体在缺氧致肾脏损伤中的作用及机制。 方法    （1）常氧（21% O₂）及缺氧（1% O₂）分别处理大鼠肾小管上皮细胞（NRK-52E）48 h，收集细胞上清液并使用高速梯度离心法分离外泌体。采用透射电镜、纳米示踪分析、Western印迹、蛋白浓度定量鉴定并比较两组外泌体的基本特性。（2）在共培养实验中，以不同浓度（1、10、50、100、300 mg/L）的常氧外泌体、缺氧外泌体分别干预脂多糖（LPS）诱导的大鼠原代腹腔巨噬细胞，使用实时荧光定量PCR与酶联免疫吸附试验（ELISA）法分别检测巨噬细胞白细胞介素6（IL-6）、肿瘤坏死因子α（TNF-α）、诱导型氮氧化物合酶（iNOS）水平；使用Western印迹法检测巨噬细胞磷酸化（p）STAT/STAT及细胞因子信号传导抑制蛋白1（SOCS1）的蛋白表达；最后，使用实时荧光定量PCR法检测常氧外泌体与缺氧外泌体中炎性反应相关微RNA（microRNA，miR）的表达差异。 结果    （1）离心得到的囊泡具有外泌体典型的结构，粒径小于150 nm，表达外泌体标志蛋白CD63，说明分离得到外泌体。缺氧对肾小管上皮细胞分泌的外泌体形态、粒径分布比例无明显影响，但提高了外泌体的分泌量。（2）缺氧外泌体相比于常氧外泌体促进了LPS诱导的M1型巨噬细胞IL-6、TNF-α、iNOS 的表达和分泌（均P＜0.01），同时提高STAT的磷酸化水平并减少SOCS1的蛋白表达（均P＜0.01）；对炎性反应相关microRNA检测发现缺氧外泌体中miR-155、miR-27a表达量较常氧外泌体明显升高（P＜0.05）。 结论    缺氧可改变外泌体的生物学功能，表现为协同促进LPS诱导的M1型巨噬细胞的表型转化，这可能是慢性肾脏病微炎性反应状态持续的原因之一。

Effect of hypoxia on exosomes in renal tubular epithelial cells

Objective    To explore the effect of hypoxia on exosomes secreted by renal tubular epithelial cells and the function of exosomes in chronic kidney diseases.    Methods    (1) The supernatant of renal tubular epithelial cells which were cultured in normoxia (21% O₂) or hypoxia(1% O₂) for 48 h was collected and centrifuged gradiently to harvest exosomes. Exosomes were identified and compared by transmission electron microscope, nanoparticle tracking analysis, Western blotting and measurement of the protein concentration. (2) Primary peritoneal macrophages of rats were co-cultured with exosomes in different concentrations (1, 10, 50, 100, 300 mg/L). The expression of interleukin-6(IL-6), tumor necrosis factor-α (TNF-α), inducible nitric oxide synthase (iNOS) in cells and supernatant were separately detected by quantitative real-time PCR (qRT-PCR) and ELISA, and the expression of phospho (p)-STAT/STAT and suppressors of cytokine signaling  1 (SOCS1) in macrophages was detected by Western blotting. At last, the expression of inflammatory microRNAs(miR) in exosomes was measured by qRT-PCR.    Results    (1) The vesicles harvested by gradient centrifugation were less than 150 nm and expressed CD63 which was characteristic of exosomes. Hypoxia had no effect on the morphology of exosomes, but stimulated their secretion. (2) Hypoxic exosomes dose-dependently improved the expression of IL-6, TNF-α, iNOS in macrophages polarized by lipopolysaccharide (LPS)  and increased the expression of p-STAT while decreased the expression of SOCS1 (P＜0.01). MicroRNAs referred to inflammation such as miR-155 and miR-27a increased in hypoxic exosomes compared to that in normoxic exosomes (P＜0.05).    Conclusions    Hypoxia makes exosomes promoted the polarization of macrophages to M1, which may account for the microinflammation in chronic kidney diseases.