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 prostatecancer-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Bar、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、48h对PC-3 细胞的 IC50分别为 5.79、6.93、4.42 μg·mL-1,而 EXO-MEL的 IC50分别为 11.95、8.58、7.67 μg·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-3mRNA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4 μg·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.
Effect of melittin-loaded exosome on prostate cancer cell and its hemolytic evaluations
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 IC50of MEL at 24/36/48h was 5.79,6.93 and 4.42 μg·mL-1 and IC50 of EXO-MEL 11.95,8.58 and 7.67 μg·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 4 μg·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.
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