VEGF和CD47双修饰外泌体靶向递送治疗沙漠干热环境热射病急性肾损伤的作用及机制研究  

Effect and mechanism of targeted delivery of VEGF and CD47 dual-modifiedexosomes in treatment of acute kidney injury induced by heat stroke in rats in the desert dry hot environment

作　　者：韩小博 邓鑫凯 李红 刘超[3] 高家威 李啸 柴雅豪 韩小丽 韩红伟 赵胤杰 杨曦[6] 张雷[1] HAN Xiaobo;DENG Xinkai;LI Hong;LIU Chao;GAO Jiawei;LI Xiao;CHAI Yahao;HAN Xiaoli;HAN Hongwei;ZHAO Yinjie;Yang Xi;ZHANG Lei(Department of Emergency Medicine,General Hospital of Xinjiang Military Command,Urumqi,Xinjiang Uygur Autonomous Region,830000;Center for Reproductive Medicine,Second Affiliated Hospital,Army Medical University(Third Military Medical University),Chongqing,400037;Department of Nephrology,Chinese PLA General Hospital,Beijing,100853,China;Department of Radiology,General Hospital of Xinjiang Military Command,Urumqi,Xinjiang Uygur Autonomous Region,830000;Department of Critical Care Medicine,General Hospital of Xinjiang Military Command,Urumqi,Xinjiang Uygur Autonomous Region,830000;Office of Scientific Research and Teaching Affairs,General Hospital of Xinjiang Military Command,Urumqi,Xinjiang Uygur Autonomous Region,830000)

机构地区：[1]新疆军区总医院急诊医学科,乌鲁木齐830000 [2]陆军军医大学第二附属医院生殖医学中心,重庆400037 [3]解放军总医院肾内科,北京100853 [4]新疆军区总医院影像科,乌鲁木齐830000 [5]新疆军区总医院重症医学科,乌鲁木齐830000 [6]新疆军区总医院科研教学办公室,乌鲁木齐830000

出　　处：《陆军军医大学学报》2024年第18期2057-2068,共12页Journal of Army Medical University

基　　金：2020年度新疆维吾尔自治区面上项目(2020D01A139)。

摘　　要：目的设计一种基于外泌体高效递送的方式,将VEGF和CD47双修饰的外泌体递送到热射病引起的肾损伤,减轻并修复肾损伤。方法构建靶向肾损伤的融合表达VEGF和CD47的质粒,转染至大鼠骨髓间充质干细胞(bone marrow derived mesenchymal stem cells,BMMSCs)后分离提取外泌体。通过透射电子显微镜及纳米颗粒跟踪分析、蛋白质印迹法等方法对外泌体进行鉴定。经大鼠尾静脉分别注射200μg DiD标记的未修饰外泌体、VEGF修饰的外泌体和VEGF-CD47双修饰的外泌体,采用小动物活体成像仪检测并分析外泌体对肾脏的靶向作用。60只SD大鼠随机分6组每组10只,sham组为空白对照组(A组,n=10),其余各组均复制热射病肾损伤模型。模型复制成功后12、24、36 h,B组给予相同剂量生理盐水;C组给予质粒空载体(Empty plasmids,Ep)组、D组给予Exos组、E组给予Exos^(VEGF)组、F组给予Exos^(VEGF-CD47)组。于3次给药治疗后第72小时取肾脏组织和血:从组织水平观察肾组织病理变化并进行损伤评分;检测血清血尿素氮(BUN)、血清肌酐(Scr)评价治疗效果;WB和qRT-PCR分析炎症介质TNF-α、NF-κB的表达水平;检测增殖调控信号分子Ki67、FGF2、pAMPK、pERK和纤维化调控分子FGF23,综合分析对增殖和抑制纤维化的作用。结果成功获取了BMMSCs和Exos^(VEGF-CD47)并进行鉴定,急性肾损伤模型动物均复制成功。Exos^(VEGF-CD47)在活体肾组织中比ExosCtrl组和Exos^(VEGF)组的荧光强度高(P<0.05)。治疗后72小时Exos^(VEGF-CD47)组,肌酐、尿素氮下降(P<0.0001);Exos^(VEGF-CD47)组Tubular casts score评分显著低于AKI+Exos组、AKI+Exos^(VEGF)组(P<0.0001);促炎因子TNF-α、NF-κB蛋白水平明显下调(P<0.0001);而Ki67、FGF2显著上调(P<0.05),FGF23显著下调(P<0.0001)。结论VEGF和CD47可以高效靶向热射病急性肾损伤,有效减轻损伤并促进修复,其机制可能与外泌体传递抑制肾组织炎症反应、促进增殖和抑制纤维�ObjectiveTo develop an effective method for delivering VEGF and CD47 double-modified exosomes to treat renal damage induced by heat stroke so as to reduce and repair renal damage.MethodsA plasmid fusion-expressing VEGF and CD47 targeting renal injury was constructed,transfected into rat bone marrow derived mesenchymal stem cells(BMMSCs),and then fusion-exosomes were isolated and extracted.Transmission electron microscopy,nanoparticle tracking analysis,and Western blotting were used to identify the obtained exosomes.Rats were intravenously injected with 200μg of DiD-labeled unmodified exosomes,VEGF-modified exosomes and VEGF-CD47 double-modified exosomes,respectively,through the tail vein,and the effects of exosomes on the kidneys were detected and analyzed using a small animal in vivo imaging instrument.A total of 60 SD rats were randomly divided into 6 groups,with 10 rats in each group,that is,blank control group(group A),heat stroke-induced renal injury model receiving PBS in 12,24 and 36 h after modelling(group B),empty plasmid group(group C),Exos group(group D),Exos^(VEGF) group(group E)and Exos^(VEGF-CD47).Kidney tissue and blood samples were collected in 72 h after 3 times of treatment.Pathological changes in kidney tissue were observed at the tissue level and the damage were scored.Changes in serum blood urea nitrogen(BUN)and serum creatinine(Scr)levels were detected to evaluate the therapeutic effect.Western blotting and qRT-PCR were used to analyze the expression of the pro-inflammatory factors TNF-αand NF-κB,the proliferation regulatory signaling molecules Ki67,FGF2,pAMPK and pERK,and the fibrosis regulatory molecule FGF23,in order to comprehensively analyze the effects on proliferation and inhibition of fibrosis.ResultsBMMSCs and Exos^(VEGF-CD47) were successfully isolated and characterized,and a rat model of acute kidney injury was effectively constructed.Higher fluorescence intensity was found in the kidney tissue of the Exos VEGF-CD47 group than the Exos-Ctrl group and Exos VEGF group(P<0.05).In

关 键 词：热射病 骨髓间充质干细胞 外泌体 VEGF CD47 急性肾损伤 

分 类 号：R594.11[医药卫生—内科学] R692.05[医药卫生—临床医学] R977.6