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目的研究肾细胞癌源性exosomes诱导人外周血单核细胞分化为髓源性抑制细胞及该细胞PD-L1分子表达情况的效应。方法超滤和蔗糖重水密度梯度离心相结合的方法分离纯化肾细胞癌源性exosomes。Ficoll密度梯度离心法分离外周血单个核细胞,贴壁法获取外周血单核细胞。倒置显微镜观察获得的单核细胞形态。流式细胞仪检测单核细胞比例及活性、经exosomes诱导的单核细胞活性情况、髓源性抑制细胞(MDSCs)及其上PD-L1分子的表达情况。结果贴壁的单核细胞形状为圆形或类圆形具少许短小伪足状突起;贴壁法获得的单核细胞比例达83.5%,活细胞比例约90.0%;髓源性抑制细胞的生成较对照组明显增加[(8.91±0.21)%vs(3.19±0.91)%,P<0.05],且该髓源性抑制细胞表达PD-L1较对照组明显增高[(88.93±8.57)%vs(44.53±5.35)%,P<0.05];体外经exosomes诱导刺激的单核细胞损伤率较对照组明显升高[(39.93±16.51)%vs(12.87±2.43)%,P<0.05]。结论肾细胞癌源性exosomes在体外能够促进单核细胞损伤,诱导单核细胞分化为髓源性抑制细胞,且该髓源性抑制细胞表达PD-L1增高,可能是其抑制T细胞免疫功能的负性调节机制之一。
Objective To investigate the effect of exosomes derived from renal cell carcinoma on the differentiation of human peripheral blood mononuclear cells into myeloid suppressor cells and the expression of PD-L1 in the cells. Methods The renal cell derived exosomes were isolated and purified by a combination of ultrafiltration and sucrose - water density gradient centrifugation. Peripheral blood mononuclear cells were isolated by Ficoll density gradient centrifugation and peripheral blood mononuclear cells were obtained by adherent method. The obtained mononuclear cells were observed under inverted microscope. The proportion and activity of monocytes, the activity of monocytes induced by exosomes and the expression of PD-L1 on myeloid derived cells (MDSCs) were detected by flow cytometry. Results Adherent mononuclear cells were round or round with a few short pseudopod-like protrusions. The proportion of mononuclear cells obtained by adherence method was 83.5% and the percentage of viable cells was about 90.0%. The myeloid-derived cells (8.91 ± 0.21)% vs (3.19 ± 0.91)%, P <0.05], and the expression of PD-L1 in myeloid derived cells was significantly higher than that in the control group [(88.93 ± 8.57)% vs 44.53 ± 5.35)%, P <0.05]. The injury rate of monocytes stimulated by exosomes in vitro was significantly higher than that of the control group [(39.93 ± 16.51)% vs (12.87 ± 2.43)%, P <0.05]. CONCLUSION: Renosome-derived exosomes can promote monocyte injury in vitro and induce monocyte differentiation into myeloid suppressor cells, and the myeloid derived inhibitory cells express PD-L1, which may be due to its inhibitory effect on T cell immune function One of the mechanisms of negative regulation.