绿色荧光蛋白标记的BHK-21细胞源性外泌体的构建及鉴定  

Construction and identification of BHK⁃21 cells⁃derived exosome labeled with green fluorescent protein

作　　者：张娟[1,2,3] 鲁明 朱海莲 何丽存 顾冉 李代英 陈俊英 潘玥[2,3] 孙强明 ZHANG Juan;LU Ming;ZHU Hai-lian;HE Li-cun;GU Ran;LI Dai-ying;CHEN Jun-ying;PAN Yue;SUN Qiang-ming(Kunming Medical University,Kunming 650500,Yunnan Province,China;不详)

机构地区：[1]昆明医科大学,云南昆明650500 [2]中国医学科学院北京协和医学院医学生物学研究所云南省重大传染病疫苗研发重点实验室,云南昆明650118 [3]云南省虫媒传染病防控研究重点实验室,云南普洱665000

出　　处：《中国生物制品学杂志》2022年第11期1331-1337,共7页Chinese Journal of Biologicals

基　　金：国家自然科学基金面上项目(31970868);云南省重大专项和重点研发计划(2019ZF004);中国医学科学院创新工程项目(2016-I2M-1-19);云南省医学领军人才项目(L-2019030).

摘　　要：目的构建稳定表达绿色荧光蛋白(green fluorescent protein,GFP)和外泌体标志蛋白CD63的BHK-21细胞,获得GFP标记的BHK-21源性的外泌体。方法将慢病毒表达质粒pCT-CD63-GFP与辅助质粒pVS-VG(Env)和pSPAX(structure)共转染293T细胞,包装重组慢病毒,测定病毒滴度后感染BHK-21细胞,72 h后用嘌呤霉素筛选稳定表达GFP的细胞。Western blot检测BHK-21细胞中CD63的表达量。取其无外泌体血清培养36 h的稳定转染株细胞上清,获得提取物,经Western blot、透射电镜及粒径分析进行鉴定。将提纯的外泌体与受体细胞BHK-21共培养6 h后,观察外泌体在细胞中的摄取情况。通过Transwell试验检测登革病毒(Dengue virus,DENV)感染后细胞外泌体分泌量的变化。结果筛选出可稳定表达GFP的BHK-21细胞株,并从细胞上清中提取了外泌体。外泌体存在特异性标志蛋白CD63和CD81;透射电镜下,外泌体呈椭圆形,具有双凹圆盘状膜结构,直径在30~200 nm之间;纳米颗粒示踪分析显示,外泌体在110.2 nm处有峰值;荧光显微镜下可见提纯的外泌体发出强烈的绿色荧光信号。提纯的外泌体与受体细胞BHK-21共培养6 h后,可在BHK-21细胞质内见到大量绿色荧光颗粒。Transwell试验发现,DENV感染后,可促进细胞释放外泌体。结论成功构建了携带GFP标记的BHK-21源性外泌体,为研究外泌体在病毒感染细胞中的信号传递提供了工具。Objective To construct BHK-21 cells stably expressing green fluorescent protein(GFP)and exosome marker protein CD63,and obtain GFP-labeled BHK-21 cells-derived exosome.Methods 293T cells were co-transfected with lentivirus expression plasmid pCT-CD63-GFP and helper plasmids pVS-VG(Env)and pSPAX(structure).The packaged recombinant lentivirus was determined for virus titer and infected to BHK-21 cells.The cells stably expressing GFP were screened with puromycin 72 h after infection and determined for expression level of CD63 by Western blot.The supernatant of stably transfected cells cultured for 36 h in exosome-free serum was collected,from which the extract was obtained and identified by Western blot,transmission electron microscopy and particle size analysis.The purified exosomes were cocultured with receptor cells BHK-21 for 6 h and observed for the uptake of exosomes.The change of exosome secretion of cells after Dengue virus(DENV)infection was determined by Transwell assay.Results BHK-21 cell lines stably expressing GFP were screened,and the exosomes were extracted from the cell supernatant,in which specific marker proteins CD63 and CD81 were observed.The exosome was elliptical in shape with a double concave disc-like membrane structure,at diameters of 30~200 nm under transmission electron microscope.Nanoparticle tracer analysis showed a peak value of exosome at 110.2 nm.Strong green fluorescence signal was observed in purified exosome under fluorescence microscope.After co-culture with purified exosomes for 6 h,a large quantity of green fluorescent particles were observed in the cytoplasm of BHK-21 cells.Transwell assay showed that DENV infection promoted the release of exosomes.Conclusion The GFP-labeled BHK-21-derived exosomes were successfully constructed,which provided a tool for study on signaling of exosomes in virusinfected cells.

关 键 词：外泌体 乳仓鼠肾细胞 绿色荧光蛋白 登革病毒 

分 类 号：R34[医药卫生—基础医学]