摘要目的 探讨氩氦冷冻胶质瘤细胞冻融产物对C57BL/6小鼠骨髓源树突状细胞(DCs)成熟的影响,以及致敏DCs诱导肿瘤杀伤效应的作用机制. 方法 采用氩氦冷冻法冻融GL261胶质瘤细胞为实验组,只插入探针不冷冻为阴性对照组,冻融等量PBS为空白对照组,采用BCA法测定冻融产物蛋白浓度;体外诱导小鼠骨髓细胞为DCs,加入各组冻融产物后观察其形态学变化.流式细胞仪检测DCs表面CD80、CD86、MHC-Ⅰ、MHC-Ⅱ的表达.ELISA检测其上清液中IL-12p70的含量;提取小鼠脾脏T细胞,与各组抗原致敏的DCs共培养后活化,流式细胞仪检测活化T细胞CD3、CD4、CD8的表达;以GL261细胞为靶细胞,以活化的T细胞即细胞毒性T细胞(CTLs)作为效应细胞,效靶比分别为10:1、50:1和100:1时CCK-8法检测CTLs对GL261的杀伤率. 结果 实验组冻融产物即肿瘤抗原浓度[(1071 ±240.94)μg/mL]高于阴性对照组[(91.42±55.75)μg/mL]及空白对照组(0μg/mL),差异有统计学意义(P＜0.05);实验组DCs有典型成熟DCs形态,表型CD80、CD86、MHC-Ⅰ、MHC-Ⅱ的表达率及IL-12p70分泌量高于阴性对照组及空白对照组,差异有统计学意义(P＜0.05);实验组CD8+T细胞表达率高,CD4+T细胞表达率低,与阴性对照组及空白对照组相比差异有统计学意义(P＜0.05);不同效靶比时实验组CTLs对GL261细胞的杀伤率显著高于阴性对照组及空白对照组,差异有统计学意义(P＜0.05). 结论 氩氦冷冻胶质瘤细胞可获取大量肿瘤抗原并促进DCs成熟,冻融产物致敏DCs后可有效负载肿瘤抗原产生抗肿瘤效应,其机制与DCs表面分子CD80、CD86、MHC-Ⅰ、MHC-Ⅱ的表达上调并诱导T细胞转化为CTLs有关.

abstractsObjective To explore the effect of Ar-He cryoablation on maturation of bone marrow-derived dendritic cells (BMDCs) in C57BL/6 mouse,and the mechanism of sensitized dendritic cells (DCs) induced tumor killing effect.Methods Ar-He cryoablation of GL261 glioma cells was used as experimental group,only inserting probe to GL261 glioma cells as negative control group and Ar-He cryoablation on the same amount of PBS as blank control group.The protein concentration of freezing and thawing products was measured.DCs were induced from BMDCs in vitro,and after rumor cell lysate was added,morphological characteristics of DCs were observed.The expressions of surface markers CD80,CD86,major histocompatibility complex-Ⅰ (MHC-Ⅰ) and MHC-Ⅱ were analyzed by flow cytometry.The IL-12p70 concentration in the supematants was detected by enzyme-linked immunosorbent assay (ELISA).T cells were extracted and co-cultured with DCs,and then,the expressions ofT cell phenotypes CD3,CD4 and CD8 were analyzed by flow cytometry.The killing rate ofT cells (choosing from sensitized DCs) to GL261 glioma cells was detected using CCK-8 method at different effect-target ratios (10:1,50:1 and 100:1).Results The protein concentration of the experimental group ([1071 ±240.94] μg/mL) was higher than that of negative control group([91.42±55.75]μg/mL) and blank control group (0 μg/mL,P＜0.05).The experimental group showed typical characteristics of mature DCs,and the expressions ofDCs phenotypes CD80,CD86,MHC-Ⅱ and MHC-Ⅱ were higher than those in the negative control group and blank control group (P＜0.05); IL-12p70 secreted by experimental group DCs was higher than that by control group (P＜0.05).There were significantly more CD8+ T cells and less CD4+ T cells in the experimental group than negative control group and blank control group (P＜0.05).The killing rate of CTLs to GL261 glioma cells in the experimental group was significantly higher than that in the negative control group and blank control group at different effect-target ratios (P＜0.05).Conclusions A large amount of tumor antigen can be obtained from GL261 glioma cells after Ar-He cryoablation which can induce maturation of DCs.After being pulsed with tumor lysate,these DCs can produce anti-tumor effect and its mechanism is related to up-regulation of DCs surface molecules CD80,CD86,MHC-Ⅰ,MHC-Ⅱ and induce T cells into cytotoxic T cells.