含蜂毒肽的前列腺癌外泌体制备及体外评价  

Preparation and in vitro evaluation of prostate cancer exosomes containing melittin

作　　者：吕立国[1] 吴巧玲[2] 黄娟 白遵光[1] 陈志强[1] 付江波 古炽明[1] 陈艳芬 LYU Li-guo;WU Qiao-ling;HUANG Juan;BAI Zun-guang;CHEN Zhi-qiang;FU Jiang-bo;GU Chi-ming;CHEN Yan-fen(Dept of Urology,Guangdong Province Hospital of Traditional Chinese Medicine,Guangzhou 510120,China;Dept of Oncology,Guangdong Province Hospital of Traditional Chinese Medicine,Guangzhou 510120,China;School of Traditional Chinese Medicine,Guangdong Pharmaceutical University,Guangzhou 510006,China)

机构地区：[1]广东省中医院泌尿外科,广东广州510120 [2]广东省中医院肿瘤科,广东广州510120 [3]广东药科大学中药学院,广东广州510006

出　　处：《中国药理学通报》2023年第2期392-399,共8页Chinese Pharmacological Bulletin

基　　金：国家区域中医(专科)诊疗中心建设专项资金资助项目(国中医药医政函[2018]205号);全国老中医药专家学术经验继承项目(国中医药办人教函[2017]125号);全国名老中医药专家传承工作室建设项目(国中医药人教涵[2022]75号);广东省中医院拔尖人才项目(No BJ2022YL04)。

摘　　要：目的构建含蜂毒肽的前列腺癌外泌体体系,并观察前列腺癌细胞对其摄取能力。方法筛选不同时间饥饿处理的细胞用于提取外泌体;应用超速离心法提取PC-3细胞的外泌体,并通过透射电镜、纳米颗粒跟踪分析仪(NTA)以及Western blot法对所提颗粒进行检测;采用反复冻融法、室温孵育法以及电穿孔法分别制备蜂毒肽外泌体体系,并利用离心法测其包封率大小;应用高效液相色谱法(HPLC)建立蜂毒肽外泌体(EXO-MEL)的含量测定方法;应用荧光倒置显微镜观察PC-3细胞、DU145细胞以及LNCaP细胞对蜂毒肽外泌体的摄取情况。结果饥饿处理24 h为最佳外泌体提取时间点;透射电镜结果显示,外泌体外形呈圆形或椭圆形,具有明显的膜性结构,直径在100 nm左右;NTA分析外泌体的试剂蛋白浓度为0.222 g·L^(-1);Western blot检测外泌体的标志蛋白结果显示Alix、CD63为阳性表达,提示采用饥饿培养PC-3细胞,结合超速离心法提取可获得外泌体。包封率检测结果表明:电穿孔法的包封率为17.51%±2.39%、反复冻融法为11.46%±1.02%、室温孵育法为3.93%±2.44%,电穿孔法包封率最大且有明显差异(P<0.05)。摄取实验表明,PC-3细胞可以有效摄取EXO-MEL,其摄取程度有一定的时间依赖性;DU145以及LNCaP细胞也能摄取EXO-MEL。结论通过饥饿处理加高速离心法可成功获取前列腺癌外泌体,通过电穿孔法制备的蜂毒肽外泌体体系可以被前列腺癌细胞摄取。Aim To prepare prostate cancer exosomes containing melittin and observe their uptake by prostate cancer cells.Methods Cells treated with starvation for different time were screened for exosome extraction.Exosomes from PC-3 cells were extracted by ultracentrifugation,and the extracted particles were examined by transmission electron microscopy,nanoparticle tracking analyzer(NTA),and Western blot.Melittin exosome system was prepared by repeated freeze-thaw method,incubation at room temperature as well as electroporation,and the size of encapsulation efficiency was measured by centrifugation.A high-performance liquid chromatography(HPLC)method was applied to assay the content of melittin exosomes(exo-mel).Fluorescence inverted microscopy was employed to evaluate the uptake of melittin exosomes by PC-3 cells,DU145 cells as well as LNCaP cells.Results The results of starvation treatment showed that 24 h starvation treatment was the optimal time point.TEM results showed that the exosomes were round or oval in shape with a distinct membranous structure,and the diameter was around 100 nm.The reagent protein concentration for NTA analysis of exosomes was 0.222 g·L^(-1).The results of Western blot for the marker proteins of exosomes showed that Alix and CD63 were positively expressed,which indicated that the exosomes could be obtained by starvation culture of PC-3 cells and ultracentrifugation.The results of entrapment efficiency showed that the entrapment efficiency of electroporation method was 17.51%±2.39%,that of repeated freeze-thaw method was 11.46%±1.02%,and that of room temperature incubation method was 3.93%±2.44%.The encapsulation efficiency of electroporation was the highest with significant difference(P<0.05).The uptake assay showed that PC-3 cells could efficiently take up exo-mel in a time-dependent manner,and DU145 cells and LNCaP cells also could take up exo-mel over time.Conclusions Exosomes can be accessed by starvation treatment and high-speed centrifugation,and the prostate cancer melittin exosome s

关 键 词：前列腺癌 外泌体 蜂毒肽 PC-3细胞 载药方式 电穿孔法 

分 类 号：R329.24[医药卫生—人体解剖和组织胚胎学] R737.25[医药卫生—基础医学] R996.3