增强型绿色荧光蛋白标记膀胱癌外泌体的构建及其体外示踪作用  

Construction of enhanced green fluorescent protein labeling bladder cancer exosomes and its tracing application in vitro

作　　者：陈辉[1] 侯爱华[1] 王培培 梅晓峰[1] CHEN Hui;HOU Aihua;WANG Peipei;MEI Xiaofeng(The First Affiliated Hospital of Henan University of Chinese Medicine,Zhengzhou 450000,China)

机构地区：[1]河南中医药大学第一附属医院,河南省郑州市450000

出　　处：《组织工程与重建外科》2022年第4期300-305,共6页Journal of Tissue Engineering and Reconstructive Surgery

摘　　要：目的 构建稳定表达CD63-增强型绿色荧光蛋白(Enhanced green fluorescent protein,EGFP)融合蛋白的膀胱癌细胞株,得到EGFP标记的膀胱癌外泌体,进行外泌体的体外示踪。方法 将CD63基因通过分子克隆插入pLVXEGFP-puro质粒中,构建CD63-EGFP融合蛋白的表达载体。通过慢病毒转染得到稳定表达CD63-EGFP融合蛋白的膀胱癌细胞株,超速离心法提取外泌体,使用透射电子显微镜、粒径分析和Western blot鉴定外泌体。通过共培养的方式检测人正常膀胱上皮细胞SV-HUC-1和人外周血单个核细胞(Peripheral blood mononuclear cell,PBMC)对膀胱癌外泌体的摄取情况。结果 构建了稳定表达CD63-EGFP融合蛋白的膀胱癌细胞株,其分泌的外泌体符合外泌体经典特征,并发现SV-HUC-1细胞和PBMC中的单核细胞可在体外摄取膀胱癌外泌体。结论 表达CD63-EGFP融合蛋白的膀胱癌细胞能分泌出荧光外泌体,可用于外泌体的体外示踪。Objective To obtain EGFP labeling exosomes of bladder cancer cells for tracking exosomes in vitro by constructing a bladder cancer cell line stably expressing CD63-EGFP fusion protein. Methods The CD63 gene was inserted into pLVX-EGFP-puro plasmid by molecular cloning to construct the expression vector of CD63-EGFP fusion protein.Bladder cancer cell lines stably expressing CD63-EGFP fusion protein were obtained by lentivirus transfection. Exosomes were extracted by ultracentrifugation and identified by transmission electron microscopy, particle size analysis and Western blot. The uptake of bladder cancer cell exosomes by SV-HUC-1 cells and peripheral blood mononuclear cell(PBMC) was detected through co-culture. Results A Bladder cancer cell line stably expressing the CD63-EGFP fusion protein was constructed, and the secreted exosomes were consistent with the classic characteristics of exosomes. SV-HUC-1 cells and Monocytes in PBMC can uptake bladder cancer cell-derived exosomes in vitro. Conclusion Bladder cancer cells expressing CD63-EGFP fusion protein can secrete fluorescent exosomes, which can be used for tracing of exosomes in vitro.

关 键 词：膀胱癌 外泌体 增强型绿色荧光蛋白 示踪 

分 类 号：Q813[生物学—生物工程]