细胞外囊泡装载阿霉素对人肝癌细胞PLC/PRF/5增殖及凋亡的影响  

Effects of doxorubicin-loaded tumor-derived extracellular vesicles on cell proliferation and apoptosis of human hepatocellular carcinoma

作　　者：骆昱煜 岳文君 骆莹[2] 高英堂[2] 张金卷[3] 刘辉[2] 王毅军[1] Luo Yuyu;Yue Wenjun;Luo Ying;Gao Yingtang;Zhang Jinjuan;Liu Hui;Wang Yijun(the Third Central Clinical College of Tianjin Medical University,Tianjin 300170,China;Tianjin Key Laboratory of Extracorporeal Life Support for Critical Diseases,Tianjin Institute of Hepatobiliary Diseases,Tianjin Third Central Hospital,Tianjin 300170,China;Department of Hepatobiliary Surgery,Tianjin Third Central Hospital,Tianjin 300170,China)

机构地区：[1]天津医科大学三中心临床学院,300170 [2]天津市第三中心医院肝胆疾病研究所,天津市重症疾病体外生命支持重点实验室,300170 [3]天津市第三中心医院肝胆外科,300170

出　　处：《中华肝胆外科杂志》2020年第4期247-252,共6页Chinese Journal of Hepatobiliary Surgery

基　　金：天津市科技计划项目(17YFZCSY01070);天津市自然科学基金(17JCYBJC26100,18JCYBJC26900);天津市卫生行业重点攻关项目(15KG114)。

摘　　要：目的探讨肿瘤细胞来源的细胞外囊泡装载阿霉素对人肝癌细胞PLC/PRF/5增殖及凋亡的影响。方法以"直接共同孵育"合成装载阿霉素的细胞外囊泡,即载药囊泡,运用透射电镜观察形态,动态光散射仪测定粒径,蛋白免疫印迹技术鉴定CD 63、HSP 70及TSG 101的表达,液相-质谱联用仪计算载药率,体外药物释放实验模拟体内载药囊泡的药物缓释,采用PKH67染色观察细胞对载药囊泡的摄取,采用MTS实验和流式细胞术检测载药囊泡对人肝癌细胞PLC/PRF/5增殖及凋亡影响。结果载药囊泡在透射电镜下呈大小不一的椭圆样双层膜结构;动态光散射仪观察直径为(115.9±5.2)nm;蛋白免疫印迹检测表达CD 63、HSP 70及TSG 101;载药囊泡包封率为0.77%;体外释放实验显示载药囊泡能缓慢释放药物;PKH67示踪实验显示16小时内肝癌细胞能够摄取载药囊泡;MTS实验结果显示,经载药囊泡处理72小时后,肝癌细胞PLC/PRF/5的活性为(54.9±3.2)%,显著低于阿霉素组的(77.7±5.4)%,差异有统计学意义(P<0.05);细胞凋亡检测结果显示,载药囊泡组处理48小时后细胞凋亡率为(47.9±7.0)%,高于阿霉素组的(38.0±1.5)%,差异有统计学意义(P<0.05)。结论载药囊泡能够抑制肝癌细胞增殖并促进其凋亡。Objective To investigate the effects of doxorubicin(Dox)-loaded tumor-derived extracellular vehicles(EVs)on cell proliferation and apoptosis of human hepatocellular carcinoma.Methods The extracellular vesicles loaded with Adriamycin(EVs-Dox)were prepared by the method of directly co-incubation.The morphology of EVs-Dox was detected by transmission electron microphotometer.The diameter of EVs-Dox was determined by dynamic light scattering(DLS).Western blotting was utilized to detect the expression of CD63,HSP 70 and TSG 101 in the EVs-Dox.The encapsulation efficiency of EVs-Dox was calculated by tandem mass spectrometry(LC-MS/MS).The drug release experiment in vitro was utilized to simulate the drug release of drug-loaded vesicles in vivo.PKH67-labeled EVs-Dox was showed cellular uptake.After treatment with EVs-Dox,MTS assay and flow cytometry assay were conducted to investigate the effects of EVs-Dox on cell proliferation and apoptosis of PLC/PRF/5.Results The EVs-Dox showed an elliptical double-layer membrane structure of different sizes under transmission electron microscope.The diameter of EVs-Dox was(115.9±5.2)nm.Western blotting data showed high expression of CD 63,HSP 70 and TSG 101 in the EVs-Dox.The encapsulation efficiency of EVs-Dox was 0.77%.The in vitro release experiment showed that the drug-loaded vesicles could release the drug slowly.PKH67-labeled EVs-Dox showed that carcinoma cells can uptake EVs-Dox within 16h.MTS assay showed that the cell viability rate of(54.9±3.2)%was significantly lower than that of in the Dox group[(77.7±5.4)%,P<0.05].EVs-Dox inhibited hepatocellular carcinoma proliferation.Flow cytometry assay showed that the apoptosis rate of EVs-Dox(47.9±7.0)%was higher than that in the Dox group[(38.0±1.5)%,P<0.05].Conclusion EVs-Dox inhibits cell proliferation and accelerates apoptosis of hepatocellular carcinoma cells.

关 键 词：癌 肝细胞 阿霉素 细胞外囊泡 增殖 凋亡 

分 类 号：R735[医药卫生—肿瘤]