载蜂毒肽外泌体对前列腺癌细胞的作用及溶血性评价  

Effect of melittin-loaded exosome on prostate cancer cell and its hemolytic evaluations

作　　者：吕立国 黄娟 吴巧玲 白遵光 陈志强 魏苑君 陈艳芬 古炽明 LYU Liguo;HUANG Juan;WU Qiaoling;BAI Zunguang;CHEN Zhiqiang;Wei Yuanjun;CHEN Yanfen;GU Chiming(Department of Urology Surgery,Second Affiliated Hospital,Guangzhou University of Chinese Medicine/Guangdong Province Hospital of Traditional Chinese Medicine,Guangdong Guang-zhou 510120,China;School of Traditional Chinese Medicine,Guangdong Pharmaceutical University,Guangdong Guang-zhou 510006,China;Department of Oncology,Second Affiliated Hospital,Guangzhou University of Chinese Medicine/Guangdong Province Hospital of Traditional Chinese Medicine,Guangdong Guang-zhou 510120,China)

机构地区：[1]广州中医药大学第二附属医院/广东省中医院,泌尿外科,广东广州510120 [2]广州中医药大学第二附属医院/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：《中国医院药学杂志》2024年第16期1843-1850,共8页Chinese Journal of Hospital Pharmacy

基　　金：国家中医药管理局全国名老中医药专家传承工作室建设项目(编号:国中医药人教函[2022]75号);广州市校(院)联合资助项目(编号:202201020350);广东省中医院拔尖人才项目(编号:BJ2022YL04)。

摘　　要：目的:研究负载蜂毒肽的外泌体体系(xosome system loaded with melittin,EXO-MEL)对人前列腺癌细胞(human prostate cancer-3,PC-3)的抑制作用及其体外溶血性。方法:利用噻唑蓝(methylthiazolyldiphenyl tetrazolium,MTT)实验、划痕实验检测EXO-MEL及蜂毒肽(MEL)对PC-3细胞增殖及迁移的影响;流式细胞术检测其对PC-3细胞周期和凋亡的影响;实时荧光定量PCR检测对Bax、Bcl-2以及caspase-3 mRNA的影响;通过红细胞溶血实验评价EXO-MEL的溶血风险。结果:MEL在24、36、48 h对PC-3细胞的IC50分别为5.79、6.93、4.42μg·mL^(–1),而EXO-MEL的IC50分别为11.95、8.58、7.67μg·mL^(–1);划痕实验显示随着MEL、EXO-MEL给药浓度的增加,PC-3细胞的迁移愈合率逐渐降低;MEL与EXO-MEL能使S期细胞的占比增大,诱导PC-3细胞凋亡;荧光定量PCR结果显示,MEL与EXO-MEL可以明显增加Bax和caspase-3 mRNA表达水平,减少Bcl-2表达;溶血实验显示MEL的溶血率高于EXO-MEL,尤其是4μg·mL^(–1)具有显著差异。结论:EXO-MEL对PC-3细胞有明显的增殖抑制、诱导凋亡、阻滞周期作用,这可能与其上调Bax和caspase-3,下调Bcl-2表达有关;与MEL相比,EXO-MEL的作用相对缓和,且在一定浓度时溶血不良反应显著减轻,提示以外泌体为载体运载MEL在泌尿系统肿瘤治疗中可能具有良好的应用前景。OBJECTIVE To explore the inhibitory effect of exosome system loaded with melittin(EXO-MEL)on human prostate cancer-3(PC-3)cell and examine its in vitro hemolysis.METHODS Methylthiazolyldiphenyl tetrazolium(MTT)and scratch tests were utilized for detecting the effect of EXO-MEL on cellular proliferation and migration.Flow cytometry was employed for detecting the effect of EXO-MEL on cellular cycle and apoptosis.Reverse transcription fluorescent quantitative poly⁃merase chain reaction(RT-qPCR)was utilized for examining the effect of EXO-MEL on mRNA expressions of Bax,Bcl-2 and caspase-3.Hemolytic risk of EXO-MEL was evaluated by erythrocyte hemolysis assay.RESULTS IC50 of MEL at 24/36/48h was 5.79,6.93 and 4.42μg·mL–1 and IC50 of EXO-MEL 11.95,8.58 and 7.67μg·mL–1 respectively.Scratch test showed that cellular migration and healing rate declined with rising concentrations of MEL and EXO-MEL.Flow cytometry showed that both MEL and EXO-MEL could enhance cellular proportion of S phase and induce cellular apoptosis.RT-qPCR indicated that MEL and EXO-MEL boosted the mRNA expressions of Bax and caspase-3 and lowered the mRNA expression of Bcl-2.Hemolysis test revealed hemolytic rate of MEL was higher than that of EXO-MEL.The difference was more pronounced at a concentration of 4μg·mL–1.CONCLUSION EXO-MEL can significantly suppress proliferation,induce apoptosis and block cell cycle in PC-3 cells through an up-regulation of Bax/caspase-3 and a down-regulation of Bcl-2 mRNA.As compared with MEL,the effect of EXO-MEL is relatively milder and hemolytic side effect declines markedly at a certain concentration.Thus using extracellular vesicles as a carrier for transporting melittin may have excellent application prospects for treating urinary system tumors.

关 键 词：蜂毒肽 外泌体 PC-3细胞 增殖 迁移 凋亡 溶血 

分 类 号：R963[医药卫生—微生物与生化药学]