【摘要】 目的探讨Lewis肺癌细胞培养上清(TCCM)对髓源性抑制细胞(MDSC)代谢和功能的影响。方法免疫磁珠法分离Lewis肺癌移植瘤小鼠脾脏MDSC,用小鼠TCCM处理MDSC 48 h后,实时荧光定量PCR检测MDSC中乳酸脱氢酶A(LDHA)及葡萄糖转运蛋白1(GLUT1)、缺氧诱导因子1α(HIF-1α)mRNA的水平,己糖激酶(HK)法检测葡萄糖浓度,乳酸检测试剂盒检测糖酵解途径终产物乳酸含量,羧基荧光素二乙酸盐琥珀酰亚胺酯(CFSE)染色结合流式细胞术检测MDSC对CD4~+ T细胞增殖的抑制作用,精氨酸酶1(ARG1)检测试剂盒检测MDSC的ARG1活性,一氧化氮试剂盒检测诱导型一氧化氮合酶(iNOS)的分泌水平。结果与未处理组相比, TCCM处理过的MDSC糖酵解途径关键酶LDHA的mRNA水平及糖酵解途径相关蛋白GLUT1、 HIF-1α的mRNA水平明显上调, MDSC分泌的免疫抑制性效应分子ARG1的活性和iNOS的分泌量显著增加, MDSC对CD4~+ T细胞增殖的抑制作用明显增强。结论 TCCM能够激活MDSC的糖酵解途径,上调其对CD4~+T细胞增殖的抑制作用及免疫抑制效应分子的释放。更多还原

【Abstract】 Objective To investigate the effect of tumor microenvironment on the metabolism and function of myeloid-derived suppressor cells(MDSCs). Methods The spleen-derived MDSCs of lung cancer xenograft mice were isolated by immunomagnetic beads. After treated by Lewis tumor cell conditioned medium(TCCM) for 48 hours, real-time PCR was used to detect the mRNA levels of lactate dehydrogenase A(LDHA), glucose transporter-1(GLUT1) and hypoxia-inducible factor 1α(HIF-1α). The hexokinase(HK) method was used to detect the concentration of glucose; the lactic acid test kit was used to detect the content of lactic acid which was the final production of glycolysis; and the 5, 6-carboxyfluorescein diacetate succinimiyl ester(CFSE) staining combined with flow cytometry was used to detect the immunosuppressive function of MDSCs on the proliferation of CD4~+ T cells. Arginase kit was used to detect arginase 1(ARG1) activity of MDSCs, and nitric oxide kit was used to detect the level of inducible nitric oxide synthase(iNOS). Results Compared with the untreated group, the mRNA levels of LDHA, GLUT1 and HIF-1α were up-regulated; the activity of ARG1 and the level of iNOS significantly increased; the immunosuppressive function of MDSCs on the proliferation of CD4~+ T cells was significantly enhanced after the treatment of TCCM. Conclusion TCCM can activate the glycolytic pathway, up-regulate the immunosuppressive function on the proliferation of CD4~+ T cells and the release of immunosuppressive effector molecule of MDSCs.更多还原