人脂肪间充质干细胞外泌体对脓毒症小鼠急性肺损伤的影响及其机制  

Effects and underlying mechanism of human adipose mesenchymal stem cells-derived exosomes on acute lung injury in septic mice

作　　者：白晓智 陶克 刘洋 郝彤 张浩 官浩 Bai Xiaozhi;Tao Ke;Liu Yang;Hao Tong;Zhang Hao;Guan Hao(Department of Burns and Cutaneous Surgery,Burn Center of PLA,the First Affiliated Hospital of Air Force Medical University,Xi'an 710032,China;Department of Wound Repair,Center for Wound Repair and Regenerative Medicine,the First Affiliated Hospital of Wenzhou Medical University,Wenzhou 325015,China)

机构地区：[1]空军军医大学第一附属医院全军烧伤中心,烧伤与皮肤外科,西安710032 [2]温州医科大学附属第一医院创面修复与再生医学中心创面修复科,温州325015

出　　处：《中华烧伤与创面修复杂志》2024年第12期1132-1142,共11页Chinese Journal of Burns And Wounds

基　　金：国家自然科学基金面上项目(82272269)。

摘　　要：目的探讨人脂肪间充质干细胞(ADSC)外泌体对脓毒症小鼠急性肺损伤的影响及其机制。方法该研究为实验研究。选取第4~5代人ADSC,采用差速超高速离心法分离并提取其上清液中的外泌体,对外泌体进行鉴定后使用。取24只成年雄性BALB/c小鼠,按照随机数字表法(分组方法下同)分成正常对照组、单纯盲肠结扎穿孔(CLP)组和CLP+ADSC外泌体组,对单纯CLP组小鼠行CLP(构建脓毒症小鼠急性肺损伤动物模型)后注射磷酸盐缓冲液,对CLP+ADSC外泌体组小鼠进行组名相应的处理,对正常对照组小鼠仅注射磷酸盐缓冲液,每组8只。术后24 h,采用苏木精-伊红染色观测小鼠肺组织形态,采用原位末端标记法检测肺组织细胞凋亡情况,采用酶联免疫吸附测定法检测小鼠血清中肿瘤坏死因子α(TNF-α)和白细胞介素1β(IL-1β)含量,使用酶标仪检测肺组织中丙二醛和超氧化物歧化酶(SOD)的含量,采用免疫荧光法检测小鼠肺组织细胞中CD86、CD206的表达。取小鼠巨噬细胞RAW264.7,分为空白对照组、单纯内毒素/脂多糖(LPS)组和LPS+ADSC外泌体组,在LPS+ADSC外泌体组和单纯LPS组细胞中分别加入LPS+ADSC外泌体、LPS进行培养,对空白对照组细胞进行常规培养。培养12 h后,采用相关试剂盒检测细胞中ATP含量、线粒体活性氧的阳性细胞百分比、线粒体膜电位情况,采用实时荧光定量反转录PCR法检测细胞中M1型极化标志因子诱导型一氧化氮合酶(iNOS)、M2型极化标志因子精氨酸酶-1(Arg1)以及炎症因子TNF-α和IL-1β的mRNA表达量。以上实验除mRNA表达量的检测样本数为3以外,其余各指标的检测样本数均为4。结果术后24 h,正常对照组小鼠肺组织结构清晰完整,无炎症细胞浸润;单纯CLP组较正常对照组小鼠的肺组织水肿明显,炎症细胞浸润现象明显,凋亡、坏死细胞明显增多;CLP+ADSC外泌体组较单纯CLP组小鼠肺组织水肿症状明显减轻,炎症Objective To explore the effects and underlying mechanism of human adipose mesenchymal stem cells(ADSC)-derived exosomes on acute lung injury in septic mice.Methods The study was an experimental study.Human ADSC of passages 4-5 were selected,and exosomes in their supernatant were isolated and extracted by differential ultracentrifugation.Exosomes were then used after identification.Twenty-four adult male BALB/c mice were selected and divided into normal control group,simple cecal ligation and puncture(CLP)group,and CLP+ADSC-exosome group according to the random number table method(the grouping method was the same below),with 8 mice in each group.The mice in simple CLP group were injected with phosphate buffer after CLP surgery(to establish an animal model of acute lung injury in septic mice),the mice in CLP+ADSC-exosome group were treated according to the corresponding group name,and the mice in normal control group were only injected with phosphate buffer.At 24 hours after surgery,the morphology of lung tissue was observed by hematoxylin-eosin staining,the apoptosis of lung tissue cells was detected by in-situ end-labeling method,the content of tumor necrosis factor-α(TNF-α)and interleukin-1β(IL-1β)in the serum of mice was detected by enzyme-linked immunosorbent assay,the content of malondialdehyde and superoxide dismutase(SOD)in lung tissue was detected by microplate reader,and the expressions of CD86 and CD206 in mouse lung tissue cells was detected by immunofluorescence method.Mouse macrophage RAW264.7 was taken and divided into blank control group,simple lipopolysaccharide(LPS)group,and LPS+ADSC-exosome group.The cells of LPS+ADSC-exosome group and simple LPS group were cultured by adding LPS+ADSC-exosome and LPS,respectively,and cells in blank control group were routinely cultured.Twelve hours after culture,the ATP content,the percentage of mitochondrial reactive oxygen species positive cells,as well as mitochondrial membrane potential in cells were detected by related detection kits.The mRNA expression

关 键 词：脓毒症 急性肺损伤 间质干细胞 外泌体 巨噬细胞 线粒体功能障碍 

分 类 号：R459.7[医药卫生—急诊医学] R563.8[医药卫生—治疗学]