膀胱癌MB49细胞exosomes对MDSCs增殖及活性的影响

Effects of exosomes derived from bladder cancer cell line MB49 on MDSCs expansion and activation

目的探讨膀胱癌MB49细胞来源的外泌体(exosomes)对MDSCs细胞扩增、活化的影响及机制。方法用超滤联合蔗糖重水密度梯度离心法分离纯化膀胱癌MB49细胞分泌的exosomes;电镜形态学分析及Western blotting鉴定;流式细胞仪检测MDSCs比例;ELISA检测VEGF和IL-6;RT-PCR和Western blotting检测cyclin D1、Arg-1 m RNA和蛋白的表达;Western blotting检测p-STAT3和p-NF-κB的变化。结果成功分离纯化得到膀胱癌MB49细胞分泌的exosomes。exosomes可促进MDSCs扩增及活化。实验组MDSCs比例明显升高;Cyclin D1及Arg-1 m RNA和蛋白的表达均上调;p-STAT3和p-NF-κB的表达也上调。结论膀胱癌MB49细胞来源的exosomes能促使MDSCs细胞扩增、活化。研究和分析MDSCs的免疫抑制机制,为疾病的治疗寻找行之有效的新靶点。

To investigate the effects of exosomes derived from bladder cancer cell line MB49 on the expansion and activation of MDSCs and explore the mechanism,exosomes derived from MB49 cells were separated and purified by ultrafiltration and sucrose gradient centrifugation,then morphology and component of the exosomes were analyzed with electron microscopy and Western blotting respectively. MDSCs proportion was detected by flow cytometry; VEGF and IL-6 produced in culture supernatants were quantified using ELISA kits. The changes of m RNA and protein expressions of cyclin D1,Arg-1 were examined using RT-PCR and Western blotting,and the changes in the protein expression of p-STAT3 and p-NF-κB were detected by Western blotting. Data showed exosomes were successfully purified by ultrafiltration and sucrose gradient centrifugation. Compared with the control cells,MDSCs treated with the exosomes showed enhanced expansion and activation. Exosomes treatment upregulated the m RNA and protein expressions of cyclin D1 and Arg-1,as well as the expressions of p-STAT3 and p-NF-κB. In conclusion,exosomes derived from MB49 can promote the expansion and activation of MDSCs.Research and analysis of MDSCs immunosuppressive mechanism would be helpful to seek new effective targets for disease treatment.