摘要
目的探讨诱导性多能干细胞来源的外泌体(induced pluripotent stem cell-derived exosome,iPSC-Exo)对脂多糖(lipopolysaccharide,LPS)刺激的小胶质细胞释放炎症因子的作用。方法将本实验室冻存的脓毒症患者尿液细胞来源iPSC复苏并培养,低温超速离心法分离iPSCExo,透射电子显微镜、免疫印迹试验和超高灵敏流式检测技术(high sensitivity flow cytometry,HSFCM)验证提取物为外泌体。LPS(100 ng/mL)刺激人小胶质细胞系HMO6细胞建立炎症反应模型,根据外泌体浓度梯度分为4组,即LPS+磷酸盐缓冲液(phosphate buffer solution,PBS)组、LPS+iPSC-Exo 10^(5)组、LPS+iPSC-Exo 10^(6)组、LPS+iPSC-Exo 10^(7)组,对照组将LPS等量替换为PBS。培养24 h后检测细胞内丙二醛(propylene glycol,MDA)浓度,RT-qPCR检测细胞内巨噬细胞炎性蛋白(macrophage inflammatory protein 2,MIP2)、肿瘤坏死因子-α(tumor necrosis factor-α,TNF-α)、白介素(interleukin,IL)-1β和IL-6的mRNA表达水平,酶联免疫吸附法测定上清液中上述炎症因子蛋白浓度。相同外泌体浓度下,组间比较采用单因素方差分析及SNK-q检验。结果提取物经透射电子显微镜观察为球形膜结构,HSFCM示提取物平均直径为(74.66±15.60)nm,浓度约为2.98×10^(10)/mL,免疫印迹试验示外泌体标志物CD63、Alix和TSG101高表达,不表达GM130。与对照组比较,LPS+PBS组细胞内MDA浓度、炎症因子(MIP2、TNF-α、IL-1β和IL-6)mRNA表达水平及蛋白浓度均明显升高(均P<0.01)。随iPSC-Exo浓度增加,细胞内MDA浓度逐渐降低(P<0.01),细胞内各炎症因子mRNA表达呈逐渐下降趋势(均P<0.05),炎症因子蛋白浓度的变化趋势与相应炎症因子mRNA基本一致。对照组炎症指标不随iPSC-Exo浓度增加而变化。结论脓毒症患者尿液细胞来源iPSC-Exo可下调LPS诱导的小胶质细胞氧化应激及炎症因子表达水平,并且抑制作用呈剂量效应性增强。
Objective To investigate the effects of induced pluripotent stem cell-derived exosome(iPSC-Exo)on releasing inflammatory factors from microglia induced by lipopolysaccharide(LPS).Methods iPSC derived from the tubular epithelial cells of sepsis encephalopathy patients were resuscitated and cultured.The iPSC-Exo was isolated by low-temperature ultracentrifugation and analyzed by transmission electron microscopy,Western blot and high sensitivity flow cytometry(HSFCM).Based on the concentration of iPSC-Exo,human microglia line HMO6 cells activated by LPS(100 ng/mL)were divided into four groups randomly:LPS+phosphate buffer solution(PBS)group,LPS+iPSC-Exo 10^(5) group,LPS+iPSC-Exo 10^(6) group and LPS+iPSC-Exo 10^(7) group.The control group was added equal PBS but not LPS.After culture for 24 h,the concentrations of malondialdehyde in cells were detected.Quantitative RT-PCR was used to measure the mRNA expression levels of macrophage inflammatory protein 2(MIP2),tumor necrosis factor-α(TNF-α),interleukin(IL)-1βand IL-6 in the cells and enzymelinked immunosorbent assay(ELISA)was used to assess the concentration of these cytokines in the supernatant.Under the same concentration of iPSC-Exo,one-way ANOVA and SNK-q test were used for comparison between groups.Results The extracts showed spherical membrane structure by transmission electron microscopy.HSFCM showed the mean diameter of the extracts was(74.66±15.60)nm and the concentration around 2.98×10^(10)/mL.Western blot analysis showed high expression of exosome markers CD63,Alix and TSG101,but not GM130.Intracellular MDA concentration and mRNA expression levels and protein concentration of MIP2,TNF-α,IL-1βand IL-6 in the LPS+PBS group were significantly higher than those in the control group(all P<0.01).With the increase of iPSC-Exo concentration,the intracellular MDA concentration decreased gradually(P<0.01),the mRNA expression levels of inflammatory factors showed a gradual downward trend(all P<0.05).Each inflammatory cytokine in the supernatant declined in a manner that was almost consistent with mRNA.Concentrations of MDA remained constant in the control group.Conclusions iPSC-Exo derived from the tubular epithelial cells of sepsis encephalopathy patients alleviate oxidative stress and inflammation effect of microglia induced by LPS,and the modulatory effect is dose-dependent.
作者
马礼秀
肖策
章智哲
詹以安
Ma Lixiu;Xiao Ce;Zhang Zhizhe;Zhan Yi'an(Department of Respiratory and Critical Care Medicine,the First Affiliated Hospital of Nanchang University,Nanchang 330006,China;China-Japan Friendship Jiangxi Hospital,Nanchang 330006,China)
出处
《中华急诊医学杂志》
CAS
CSCD
北大核心
2023年第1期52-58,共7页
Chinese Journal of Emergency Medicine
基金
国家自然科学基金(82060345)
江西省自然科学基金(20192BAB205057)。
关键词
外泌体
诱导性多能干细胞
小胶质细胞
脓毒症脑损伤
脂多糖
炎症因子
Exosome
Induced pluripotent stem cell
Microglia
Sepsis encephalopathy
Lipopolysaccharide
Inflammatory factor