骨肉瘤干细胞来源外泌体对T细胞增殖及分化的影响

Effect of exosomes derived from osteosarcoma stem cells on proliferation and differentiation of T cells

目的探讨骨肉瘤干细胞(osteosarcoma stem cells,OSCs)来源的外泌体对T细胞增殖及分化的影响及其相关作用机制。方法采用无血清悬浮培养获得OSCs,并利用干细胞标志物流式分选技术进行鉴定。试剂盒提取骨肉瘤干细胞分泌的外泌体(OSCs-exo),利用透射电镜、粒径分析及Western blot实验进行鉴定。将OSCs-exo与PBMC进行共培养,CFSE染色及细胞流式实验检测其对T细胞增殖的影响。Western blot实验检测ERK信号通路中ERK蛋白的磷酸化改变。免疫磁珠分选CD4+T细胞,并将其与OSCs-exo在不同CD4+T细胞亚群诱导分化液中进行共培养,细胞流式实验检测OSCs-exo对后者分化的影响。Western blot实验检测STAT信号通路中磷酸化STAT1、STAT3、STAT5蛋白的表达。结果成功分离获得了OSCs CD133+MG-63,透视电镜、粒径分析及Western blot实验结果表明OSCs-exo为直径在30~100 nm的圆形或椭圆形囊泡结构,其高表达外泌体标志性蛋白CD63、HSP70。CFSE染色及细胞流式实验及Western blot实验结果显示OSCs-exo可能通过抑制ERK蛋白磷酸化抑制CD3+、CD4+、CD8+T细胞的增殖,通过抑制磷酸化STAT1、STAT3蛋白的表达抑制CD4+T细胞向Th1、Th17并促进其向Th2、Treg细胞的分化。结论骨肉瘤干细胞来源的外泌体能够通过下调ERK蛋白的磷酸化抑制T细胞的增殖,并降低磷酸化STAT1与STAT3蛋白的表达以诱导CD4+T细胞向Th2、Treg细胞分化,而抑制其向Th1、Th17的分化,从而下调细胞免疫功能,促进肿瘤的进展。

Objective To investigate the effect of exosomes derived from osteosarcoma stem cells(OSCs)on the proliferation and differentiation of T cells and to explore its mechanism.Methods OSCs were obtained using serum-free suspension culture and identified using stem cell marker logistics separation technology.The exosomes(OSCs-exo)secreted by OSCs were extracted using an exosome extraction kit and identified using transmission electron microscopy(TEM),particle size analysis,and Western blot.Coculture of OSCs-exo with peripheral blood mononuclear cells,carboxyfluorescein succinimidyl ester(CFSE)staining,and flow cytometry were used to detect the effect on T-cell proliferation.Western blot was used to detect the phosphorylation of extracellular signal-regulated kinase(ERK)protein in the ERK signaling pathway.Immunomagnetic beads were used to separate CD4+T cells and coculture them with OSCs-exo in the differentiation medium induced by different CD4+T cell subsets.Western blot was used to detect the expression of phosphorylated STAT1,STAT3,and STAT5 proteins in the STAT signaling pathway.Results OSCs CD133+MG-63 was successfully isolated.The result of TEM,particle size analysis,and Western blot showed that OSCs-exo is a round or oval vesicle with a diameter of 30-100 nm.The CFSE staining,flow cytometry,and Western blot result showed that OSCs-exo inhibits the proliferation of CD3+,CD4+,and CD8+T cells by inhibiting the phosphorylation of ERK protein,and inhibits the differentiation of CD4+T cells to Th1,Th17,Th2,and Treg cells by inhibiting the expression of phosphorylated STAT1 and STAT3 protein.Conclusions Exosomes derived from OSCs can inhibit the proliferation of T cells by downregulating the phosphorylation of ERK protein and reducing the expression of phosphorylated STAT1 and STAT3 protein to induce CD4+T cells to differentiate into Th2 and Treg cells.They can also inhibit the differentiation of CD4+T cells into Th1 and Th17 cells to downregulate cellular immune function and promote tumor progress.