间充质干细胞来源的细胞外囊泡对放射性肝损伤的保护作用及其机制研究  

Protective effect and mechanism of mesenchymal stem cell-derived extracellular vesicle on radiation-induced liver injury

作　　者：陈池 萧家麒 隋昕 张英才[1] Chen Chi;Xiao Jiaqi;Sui Xin;Zhang Yingcai(Department of Hepatic Surgery,Liver Transplantation Center,the Third Affiliated Hospital of Sun Yat-sen University,Guangzhou 510630,China)

机构地区：[1]中山大学附属第三医院肝脏外科暨肝移植中心,广州510630

出　　处：《器官移植》2023年第3期411-419,共9页Organ Transplantation

基　　金：国家自然科学基金(81970567、82270689);广东省自然科学基金(2021A1515010571);广州市科技计划项目(202201010815)。

摘　　要：目的探讨间充质干细胞来源的细胞外囊泡(MSC-EV)对照射诱导的小鼠放射性肝损伤和肝细胞系损伤的保护作用及相关机制。方法分别将C57BL/6小鼠随机分为空白组、造模组和MSC-EV治疗组(治疗组),每组9只;将AML12细胞随机分为对照组、照射组和MSC-EV干预组(干预组)。分别通过一次性15 Gy和6 Gy X线照射建立动物和细胞放射性损伤模型,照射后48 h取小鼠的肝组织与血清进行相关实验,照射后15 h对细胞进行相关检测。检测血清丙氨酸转氨酶(ALT)、天冬氨酸转氨酶(AST)水平,肝组织和细胞丙二醛(MDA)含量,实时荧光定量逆转录聚合酶链反应(RT-qPCR)检测白细胞介素(IL)-1β、IL-6、转化生长因子(TGF)-β、CXC趋化因子配体(CXCL)10信使RNA(mRNA)相对表达量,肝组织苏木素-伊红(HE)染色进行肝组织病理损伤评分,脱氧核糖核酸末端转移酶介导的dUTP缺口末端标记(TUNEL)染色法、碘化丙啶(PI)染色法分别检测肝组织、细胞凋亡情况,蛋白质印迹法测定谷胱甘肽过氧化酶(GPX)4、铁死亡抑制蛋白(FSP)1的蛋白表达情况,二氢乙啶(DHE)染色检测活性氧簇(ROS)产生水平,并检测线粒体通透性转换孔(mPTP)荧光强度。结果与空白组比较,造模组小鼠血清AST、ALT水平均增高,小鼠肝组织IL-1β、TGF-β和CXCL10 mRNA相对表达量均增加,MDA含量增加,肝损伤评分升高,细胞凋亡率增加,细胞内ROS水平升高,小鼠肝组织GPX4、FSP1蛋白相对表达量下降,差异均有统计学意义(均为P<0.05);与造模组比较,治疗组小鼠血清AST、ALT水平均降低,小鼠肝组织IL-1β、TGF-β和CXCL10 mRNA相对表达量均下降,MDA含量减少,肝损伤评分降低,细胞凋亡率下降,细胞内ROS水平降低,小鼠肝组织GPX4、FSP1蛋白相对表达量增加,差异均有统计学意义(均为P<0.05)。与对照组比较,照射组细胞凋亡率增加,细胞内ROS水平升高,mPTP荧光强度减弱,IL-1β、TGF-β、IL-6的mRNA相对表达量均Objective To evaluate the protective effect and the underlying mechanism of mesenchymal stem cell-derived extracellular vesicle(MSC-EV)on radiation-induced liver injury and liver cell line injury in mouse models.Methods C57BL/6 mice were randomly divided into the blank group,model group and MSC-EV treatment group(treatment group),with 9 mice in each group.AML12 cells were randomly divided into the control group,irradiation group and MSC-EV intervention group(intervention group).Animal and cell models with radiation-induced injury were established by one-time 15 Gy and 6 Gy X-ray irradiation,respectively.At 48 h after irradiation,liver tissues and serum samples of mice were collected and prepared for subsequent experiments.At 15 h post-irradiation,cell experiment was carried out.Serum levels of alanine aminotransferase(ALT),aspartate aminotransferase(AST)and content of malondialdehyde(MDA)in liver tissues and cells were measured.The relative expression levels of interleukin(IL)-1β,IL-6,transforming growth factor(TGF)-βand CXC chemokine ligand(CXCL)10 messenger RNA(mRNA)were detected by real-time fluorescent quantitative polymerase chain reaction(RT-qPCR).Liver tissues were prepared for hematoxylin-eosin(HE)staining to calculate liver pathological injury score.The apoptosis of liver tissues and cells was assessed by terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling(TUNEL)and propidiumiodide(PI)staining,respectively.The expression levels of glutathione peroxidase 4(GPX4)and ferroptosis suppressor protein 1(FSP1)proteins were detected by Western blot.The production level of reactive oxygen species(ROS)was detected by dihydroethidine(DHE)staining.The fluorescence intensity of mitochondrial permeability transition pore(mPTP)was determined.Results Compared with the blank group,serum levels of AST and ALT were up-regulated,and the relative expression levels of IL-1β,TGF-βand CXCL10 mRNA in the mouse liver tissues were up-regulated,and MDA content was increased,liver injury score was elevated,cell

关 键 词：细胞外囊泡 放射治疗 放射性肝损伤 铁死亡 氧化应激 脂质过氧化 炎症 线粒体通透性转换孔 

分 类 号：R575[医药卫生—消化系统] R818.74[医药卫生—内科学]