溴隐亭促进大鼠泌乳素瘤MMQ细胞分泌外泌体以及对细胞耐药性的影响

Bromocriptine promotes secretion of exosomes and drug resistance of rat prolactinoma MMQ cells in vitro

目的:探讨溴隐亭在促进大鼠泌乳素瘤MMQ细胞分泌外泌体,及其外泌体在MMQ细胞耐药中的作用。方法:采用CCK-8法检测不同浓度的溴隐亭对MMQ细胞增殖的影响,并计算半数抑制浓度(half maximal inhibitory concentration,IC50)值。MMQ细胞在溴隐亭中暴露48 h后,采用差速离心法分离收集外泌体;在透射电子显微镜下观察外泌体形态和大小,纳米颗粒跟踪分析仪检测外泌体粒径,蛋白质印迹法检测外泌体表面标志物CD63的表达情况。用PKH67标记外泌体,在激光共聚焦显微镜下观察外泌体被MMQ细胞摄取的情况;采用CCK-8法检测外泌体被摄取后,MMQ细胞对溴隐亭敏感性的改变。实时荧光定量PCR法检测外泌体处理后对MMQ细胞中PR结构域锌指蛋白2(PR domain zinc nger protein 2,PRDM2)mRNA表达的影响;蛋白质印迹法检测外泌体处理后对MMQ细胞中PRDM2、多巴胺2受体(dopamine 2 receptor,D2R)和细胞外信号调节激酶1/2(extracellular signal-regulated kinase 1/2,ERK1/2)以及磷酸化ERK1/2(phospho-ERK1/2,p-ERK1/2)表达水平的影响。结果:溴隐亭对MMQ细胞的IC50值为(52.91±1.48)μmol/L;与未用溴隐亭处理的MMQ细胞所分泌的外泌体相比,溴隐亭处理MMQ细胞所分泌的外泌体颗粒数和蛋白质含量均显著增加(P值均<0.01),但粒径没有变化。与MMQ细胞相比,仅有外泌体上有CD63的表达。溴隐亭处理MMQ细胞所分泌的外泌体可以被MMQ细胞所摄取;MMQ细胞摄取外泌体后,溴隐亭对MMQ细胞的IC50值为(73.74±1.17)μmol/L,较未摄取外泌体的MMQ细胞明显提高(P<0.01);MMQ细胞摄取外泌体后,MMQ细胞中PRDM2 mRNA和蛋白的表达水平明显下调(P值均<0.05),D2R蛋白的表达水平也明显降低(P<0.05),p-ERK1/2蛋白的表达水平升高(P<0.05)。结论:溴隐亭可促进大鼠泌乳素瘤MMQ细胞分泌外泌体,并且在溴隐亭治疗时所产生的外泌体可通过在肿瘤细胞间传递信息,从而使MMQ细胞获得耐药性。

Objective:To investigate the effect of bromocriptine on the secretion of exosomes and the role of exosomes in drug resistance of rat prolactinoma MMQ cells.Methods:The effect of different concentrations of bromocriptine on the proliferation of MMQ cells was detected by CCK-8 assay,and the half maximal inhibitory concentration(IC50)was calculated.MMQ cells were exposed to bromocriptine for 48 h,then the exosomes were isolated by differential centrifugation.The morphology of exosomes was observed by transmission electron microscopy,the particle size of exosomes was analyzed by nanoparticle tracking analysis(NTA),and the expression of exosomal marker CD63 was detected by Western blotting.MMQ cells uptaking PKH67-labeled exosomes were observed by laser confocal microscopy.Then the sensitivity of MMQ cells to bromocriptine was tested again by CCK-8 method.The expression level of PR domain zinc finger protein 2(PRDM2)mRNA in MMQ cells treated with exosomes was detected by real-time fluorescent quantitative PCR.The expression levels of PRDM2,dopamine 2 receptor(D2R),extracellular signal-regulated kinase 1/2(ERK1/2)and phospho-ERK1/2(p-ERK1/2)proteins in MMQ cells treated with exosomes were determined by Western blotting.Results:The IC50 of bromocriptine on MMQ cells was(52.91±1.48)μmol/L.Compared with the exosomes collected from MMQ cells not treated with bromocriptine,the particles and proteins in exosomes collected from MMQ cells treated with 50μmol/L bromocriptine were significantly increased(both P<0.01).The expression of CD63 was only observed in the exosomes.The exosomes collected from MMQ cells treated with bromocriptine were uptaken by MMQ cells.After the exosomes were absorbed by MMQ cells,the IC50 of bromocriptine[(73.74±1.17)μmol/L]was increased on MMQ cells(P<0.01).In exosome-treated MMQ cells,the expression levels of PRDM2 mRNA and protein(both P<0.05)and D2R protein(P<0.05)were down-regulated,while the expression level of p-ERK1/2 was higher than that in the control group(P<0.05).Conclusion:Bromocriptine can promote the secretion of exosomes in MMQ cells,and the exosomes may be involved in the transmission of information through intercellular communication of tumors,which can make MMQ cells to obtain drug resistance.