不同剂量人脐带间充质干细胞外泌体对严重烧伤大鼠肠屏障损伤修复疗效的对比研究  

Comparative study of the effect of different doses of human umbilical cord mesenchymal stem cells exosomes on intestinal barrier injury in severely burned rats

作　　者：康雨欣 韩绍芳 柴伟[3] 张勇[2] 张倞 余华庆 钱培培 王泽 刘玲英 Kang Yuxin;Han Shaofang;Chai Wei;Zhang Yong;Zhang Jing;Yu Huaqing;Qian Peipei;Wang Ze;Liu Lingying(Basic Medical College,Hebei North University,Zhangjiakou 075031,China;Department of Nutrition,the Fourth Medical Center,Chinese PLA General Hospital,Beijing 100037,China;Department of Orthopedics,the Fourth Medical Center,Chinese PLA General Hospital,Beijing 100037,China;School of Basic Medical Sciences,Inner Mongolia Medical University,Hohhot 010107,China;Department of Critical Care Medicine,the Fourth Medical Center,Chinese PLA General Hospital,Beijing 100037,China)

机构地区：[1]河北北方学院基础医学院,张家口075031 [2]解放军总医院第四医学中心营养科,北京100037 [3]解放军总医院第四医学中心骨科医学部,北京100037 [4]内蒙古医科大学基础医学院,呼和浩特010107 [5]解放军总医院第四医学中心重症医学科,北京100037

出　　处：《中华医学杂志》2024年第45期4153-4161,共9页National Medical Journal of China

基　　金：解放军总医院青年自主创新科学基金项目(22QNCZ031);国家自然科学基金联合基金(U22A20355);国家自然科学基金青年项目(81701900)。

摘　　要：目的探讨3个不同剂量人脐带间充质干细胞外泌体(hucMSC‑EXO)对严重烧伤大鼠肠屏障结构损伤和功能障碍的修复疗效,明确hucMSC‑EXO修复治疗肠屏障损伤的最佳剂量。方法使用外泌体提取纯化试剂盒分离提取hucMSC‑EXO,并鉴定。选取30只无特定病原体级6~8周龄雄性Wistar大鼠,按照随机数字表法分为5组(n=6):假伤组、烧伤组、烧伤+100μg hucMSC‑EXO组(烧伤+EXO100)、烧伤+200μg hucMSC‑EXO组(烧伤+EXO200)、烧伤+400μg hucMSC‑EXO组(烧伤+EXO400)。大鼠经94℃水浴,背部12 s和腹部6 s致伤,建立总体表面积(TBSA)50%的Ⅲ度烧伤动物模型,假伤组大鼠相同条件下经37℃水浴。致伤后第1、3、5天,假伤组和烧伤组分别予以腹腔注射磷酸盐缓冲液0.5 ml,而烧伤+EXO100、烧伤+EXO200及烧伤+EXO400则分别予以腹腔注射100、200、400μg/0.5 ml的hucMSC‑EXO。每日观察大鼠活动状况并记录体重。致伤后第7天,采集各组大鼠小肠组织和血清,苏木精‑伊红(HE)染色观察小肠组织的病理变化,酶联免疫吸附实验检测小肠组织中炎症因子肿瘤坏死因子‑α(TNF‑α)、白细胞介素‑1β(IL‑1β)、IL‑6、IL‑8、IL‑10以及细菌脂多糖(LPS)的水平,肠道屏障功能生化指标分析系统检测血清中二胺氧化酶、D‑乳酸和细菌内毒素的水平。结果hucMSC‑EXO形态呈圆形或椭圆形,大小均匀,直径峰值约为100 nm,阳性表达CD63和肿瘤易感基因101(TSG101)。假伤组大鼠体重增长约(6.3±1.2)g/d,烧伤组体重在伤后第1天显著下降,随后以(1.6±0.5)g/d的速率缓慢增长;而烧伤+EXO100、200、400组大鼠体重增长速度为(2.9±1.1)g/d,其中伤后第7天烧伤+EXO200、400组体重高于烧伤组和烧伤+EXO100组(均P<0.05)。HE染色显示,烧伤组小肠组织(十二指肠、空肠、回肠)绒毛高度[(711±35)、(526±25)、(418±33)μm]明显缩短,结构损伤严重,而EXO处理组的小肠组织结构和绒毛高度均有所恢复,�Objective To explore the therapeutic efficacies of three different doses of human umbilical cord mesenchymal stem cell exosomes(hucMSC‑EXO)on the injury of intestinal barrier structure and dysfunction in severely burned rats,and to identify the optimal dose of hucMSC‑EXO for the repair of intestinal barrier injury.Methods The hucMSC‑EXO was isolated and identified by using an exosome extraction and purification kit.A total of 30 specific pathogen free(SPF)male Wistar rats(aged 6‑8 weeks)were selected,and were randomly divided into five groups(n=6)using a random number table:sham group,burn group,burn+100μg hucMSC‑EXO group(Burn+EXO100),burn+200μg hucMSC‑EXO group(Burn+EXO200),and burn+400μg hucMSC‑EXO group(Burn+EXO400).The rats were immersed in 94℃water,with the dorsal area exposed for 12 seconds and the ventral area for 6 seconds,to establish a 50%total body surface area(TBSA)third‑degree burn model.The sham group rats were subjected under the same condition but with a 37℃water bath.On day 1,3,and 5 post‑burn,the rats in sham group and burn group received an intraperitoneal injection of 0.5 ml phosphate buffered solution,and those in Burn+EXO100,Burn+EXO200,and Burn+EXO400 groups received intraperitoneal injections of 100,200,and 400μg/0.5 ml hucMSC‑EXO,respectively.The activity of rats was observed and the weight was recorded daily.On day 7,the small intestine tissues and serum of the rats were collected.Hematoxylin‑Eosin(HE)staining was used to observe the pathological changes of the small intestinal tissues,and the levels of inflammatory factors,such as tumor necrosis factor(TNF)‑α,interleukin(IL)‑1β,IL‑6,IL‑8,IL‑10 and lipopolysaccharide(LPS)of small intestine tissues were detected by enzyme‑linked immunosorbent assay(ELISA).The levels of diamine oxidase,D‑lactic acid and bacterial endotoxin in serum were detected by intestinal barrier function biochemical analysis system.Results The morphology of hucMSC‑EXO was observed to be round or oval,with uniform size a

关 键 词：烧伤 人脐带间充质干细胞外泌体 大鼠 肠屏障 炎症因子 剂量 

分 类 号：R644[医药卫生—外科学]