多房棘球蚴源性外泌体对巨噬细胞极化的影响  

Effect of exosomes derived from Echinococcus multilocularis on macrophage polarization:A preliminary study

作　　者：冶赓博 陈功富 崔紫烟 吴俊杰 黄登亮 尹凤娇 王志鑫[1,2] 于文昊 孔繁玉 樊海宁 任利[1,2] YE Gengbo;CHEN Gongfu;CUI Ziyan;WU Junjie;HUANG Dengliang;YIN Fengjiao;WANG Zhixin;YU Wenhao;KONG Fanyu;FAN Haining;REN Li(Department of Hepatopancreatobiliary Surgery,The Affiliated Hospital of Qinghai University,Xining 810001,China;Qinghai Province Key Laboratory of Hydatid Disease Research,Xining 810001,China)

机构地区：[1]青海大学附属医院肝胆胰外科,西宁810001 [2]青海省包虫病研究重点实验室,西宁810001

出　　处：《临床肝胆病杂志》2023年第4期876-884,共9页Journal of Clinical Hepatology

基　　金：青海省科技厅项目(2019-ZJ-7031)。

摘　　要：目的探究不同时间及浓度多房棘球蚴源性外泌体对巨噬细胞极化的影响。方法从60只造模BALB/c小鼠中随机选取4只,应用7.0T MRI观察腹腔病灶生长情况;解剖造模小鼠,通过腹腔病灶提取原头节进行体外培养,超速离心法从培养上清中提取外泌体,透射电镜及蛋白质免疫印迹法鉴定外泌体表征。将未加外泌体处理的巨噬细胞单独培养组设为对照组,不同浓度多房棘球蚴来源外泌体与巨噬细胞共培养组设为实验组(10μg/mL组、50μg/mL组),分别共培养48 h和72 h,显微镜下观察巨噬细胞形态变化。通过流式细胞术和酶联免疫吸附实验(ELISA)检测极化状态。符合正态分布的计量资料多组间比较采用单因素方差分析,进一步两两比较采用LSD-t检验。结果7.0T MRI显示小鼠腹腔内弥漫分布、大小不等的病灶形成;多房棘球蚴源性外泌体直径100 nm左右,呈杯型或茶托型,其表面标志物CD9、TSG101和CD63表达阳性。共培养后,实验组大部分细胞拉长,形态不规则,主要呈多角形;流式细胞术检测发现,共培养48 h,对照组CD16/32、CD206、CD369阳性率分别为(99.53±0.06)%、(90.27±0.21)%、(2.40±0.20)%;与对照组相比,除10μg/mL外泌体组CD369阳性率[(0.80±0.00)%]降低(P<0.05),其余组别CD16/32、CD206、CD369阳性率均明显升高(P值均<0.0001);共培养72 h,对照组CD16/32、CD206、CD369阳性率分别为(99.67±0.06)%、(85.47±0.55)%、(6.60±0.20)%,实验组CD16/32、CD206、CD369阳性率较对照组均明显升高(P值均<0.05)。ELISA结果示:共培养48 h,对照组IL-6及TNFα水平分别为(58.53±15.52)pg/mL、(320.70±5.30)pg/mL,实验组外泌体浓度为50μg/mL时IL-6[(98.81±15.55)pg/mL]较对照组升高(P<0.05);共培养72 h,对照组IL-6及TNFα水平分别为(76.22±9.68)pg/mL、(323.90±87.37)pg/mL,当外泌体浓度为10μg/mL时TNFα水平[(164.20±14.17)pg/mL]较对照组明显下降(P<0.05);当外泌体浓度为50μg/mL时IL-6水平[(99.52±8.35)pg/Objective To investigate the effect of exosomes derived from Echinococcus multilocularis on macrophage polarization after treatment for different durations and concentrations.Methods A total of 60 BALB/c mice were used for modeling,among which 4 mice were selected to observe the growth of abdominal lesions on 7.0T MRI.The mice for modeling were dissected,and the protoscoleces was taken from the abdominal lesion and cultured in vitro;ultracentrifugation was used to extract the exosomes from the supernatant,and transmission electron microscopy and Western blotting were used for the characterization of exosomes.The macrophages without exosome treatment were established as control group,and the macrophages co-cultured with different concentrations of exosomes derived from Echinococcus multilocularis were established as experimental group(10μg/mL group and 50μg/mL group)and were cultured for 48 and 72 hours.The morphological changes of macrophages were observed under a microscope,and flow cytometry and ELISA were used to observe polarization state.A one-way analysis of variance was used for comparison of normally distributed continuous data between multiple groups,and the least significant difference t-test was used for further comparison between two groups.Results The results of 7.0T MRI showed the formation of diffuse lesions with different sizes in the abdominal cavity of mice,and the exosomes derived from Echinococcus multilocularis were approximately 100 nm in diameter and were cup-shaped or saucer-shaped,with the positive expression of the surface markers CD9,TSG101,and CD63.After co-culture,most of the cells in the experimental group were elongated with an irregular and polygonal shape.Flow cytometry showed that after 48 hours of co-culture,the positive rates of CD16/32,CD206,and CD369 in the control group were 99.53%±0.06%,90.27%±0.21%,and 2.40%±0.20%,respectively;compared with the control group,except that the 10μg/mL exosome group had a significant reduction in the positive rate of CD369(0.80%±0.00%)(

关 键 词：棘球蚴病 肝 外泌体 巨噬细胞 小鼠 近交BALB C 

分 类 号：R532.32[医药卫生—内科学]