脐带间充质干细胞及其条件培养基对脓毒症小鼠肠道菌群的调节作用  

Regulatory effects of umbilical cord mesenchymal stem cells and their conditioned medium on gut microbiota of septic mice

作　　者：范宇萱 余追[2] 林路路 徐志红[1] 刘韩慧 李银萍[1] Fan Yuxuan;Yu Zhui;Lin Lulu;Xu Zhihong;Liu Hanhui;Li Yinping(Taikang Medical School(School of Basic Medical Sciences),Wuhan University,Wuhan 430071,Hubei,China;Department of Critical Care Medicine,Renmin Hospital of Wuhan University,Wuhan 430060,Hubei,China)

机构地区：[1]武汉大学泰康医学院(基础医学院),湖北武汉430071 [2]武汉大学人民医院重症医学科,湖北武汉430060

出　　处：《中华危重病急救医学》2023年第1期43-50,共8页Chinese Critical Care Medicine

基　　金：国家自然科学基金(82172176,81771707)。

摘　　要：目的:观察并比较脐带间充质干细胞(MSC)及其条件培养基(MSC-CM)对脓毒症小鼠肠道菌群的调节作用。方法:将28只6~8周龄雌性C57BL/6J小鼠随机分为假手术组(Sham组)、脓毒症模型组(CLP组)、脓毒症+MSC干预组(CLP+MSC组)、脓毒症+MSC-CM干预组(CLP+MSC-CM组),每组7只。通过盲肠结扎穿孔术(CLP)构建脓毒症小鼠模型;Sham组不进行盲肠结扎和穿孔,余操作同CLP组。CLP+MSC组和CLP+MSC-CM组术后6 h分别经腹腔注射0.2 mL 1×10^(6) MSC和0.2 mL浓缩MSC-CM;Sham组和CLP组腹腔注射0.2 mL无菌磷酸盐缓冲液(PBS)。通过组织苏木素-伊红(HE)染色及结肠长度评估组织病理学改变,通过酶联免疫吸附试验(ELISA)检测血清炎症因子水平,通过流式细胞术检测腹腔巨噬细胞极化表型,通过16S rRNA测序分析肠道菌群。结果:与Sham组比较,CLP组小鼠肺组织出现显著炎症病变,结肠长度明显缩短(cm:6.00±0.26比7.11±0.09),血清促炎因子白细胞介素-1β(IL-1β)水平显著升高(ng/L:432.70±17.68比353.70±17.01),腹腔巨噬细胞F4/80+比例显著升高〔(68.25±3.41)%比(50.84±4.98)%〕,抗炎型巨噬细胞F4/80+CD206+比例显著降低〔(45.25±6.75)%比(66.66±3.36)%〕,肠道菌群α多样性sobs指数显著降低(118.50±23.25比255.70±6.87),物种组成结构改变,与转录、次生代谢物生物合成及运输与代谢、糖类运输与代谢、信号转导功能相关的菌群的相对丰度显著降低(均P<0.05)。与CLP组比较,MSC和MSC-CM干预可不同程度地减轻小鼠肺组织和结肠组织病理损伤,延长结肠长度(cm:6.53±0.27、6.87±0.18比6.00±0.26),降低血清IL-1β的水平(ng/L:382.10±16.93、343.20±23.61比432.70±17.68),降低腹腔巨噬细胞F4/80+比例〔(47.65±3.93)%、(48.68±2.51)%比(68.25±3.41)%〕,增加腹腔抗炎型巨噬细胞F4/80+CD206+比例〔(52.73±5.02)%、(66.38±4.73)%比(45.25±6.75)%〕,上调肠道菌群α多样性sobs指数(182.50±16.35、214.00±31.18比118.50±23.25),且以MSC-CM�Objective To investigate and compare the regulatory effects of umbilical cord mesenchymal stem cells(MSC)and their conditioned medium(MSC-CM)on gut microbiota of septic mice.Methods Twenty-eight six-to-eight-week-old female C57BL/6J mice were randomly divided into sham operation group(Sham group),sepsis model group(CLP group),sepsis+MSC treatment group(CLP+MSC group)and sepsis+MSC-CM treatment group(CLP+MSC-CM group),with seven mice in each group.The septic mouse model was established by cecal ligation and puncture(CLP).In Sham group,CLP were not performed,and other operations were the same as CLP group.Mice in the CLP+MSC group and CLP+MSC-CM group received 0.2 mL 1×10^(6) MSC or 0.2 mL concentrated MSC-CM via intraperitoneal injection 6 hours after CLP,respectively.Sham group and CLP group were given 0.2 mL sterile phosphate buffer saline(PBS)via intraperitoneal injection.Histopathological changes were evaluated by hematoxylin-eosin(HE)staining and colon length.Levels of inflammatory factors in serum were detected by enzyme-linked immunosorbent assay(ELISA).Phenotype of peritoneal macrophages was analyzed by flow cytometry,and the gut microbiota was analyzed via 16S rRNA sequencing.Results Compared with Sham group,significant inflammatory injury in lung and colon was observed,and shorter colon was detected in CLP group(cm:6.00±0.26 vs.7.11±0.09),the level of inflammatory cytokine interleukin-1β(IL-1β)in serum was significantly increased(ng/L:432.70±17.68 vs.353.70±17.01),the proportion of F4/80+peritoneal macrophages was increased[(68.25±3.41)%vs.(50.84±4.98)%],while the ratio of F4/80+CD206+anti-inflammatory peritoneal macrophages was decreased[(45.25±6.75)%vs.(66.66±3.36)%].Theαdiversity sobs index of gut microbiota was downregulated significantly(118.50±23.25 vs.255.70±6.87),the structure of species composition was altered,and the relative abundance of functional gut microbiota related to transcription,secondary metabolites biosynthesis,transport and catabolism,carbohydrate transport and metabo

关 键 词：间充质干细胞 条件培养基 脓毒症 巨噬细胞 肠道菌群 

分 类 号：R459.7[医药卫生—急诊医学]