结直肠癌细胞所致免疫抑制及免疫抑制分子分泌模式的研究

Models of immunosuppression and immunosuppressor secretion from Colon26 colorectal tumor cells

目的探讨结直肠癌细胞分泌的免疫抑制物质及抑制免疫功能的模式。方法制备结直肠癌Colon26细胞培养上清,采用MTT法检测其对T细胞转化和NK细胞的影响,直接免疫荧光FCM法检测对IL-2Rα、CD3ε+ζ+及CD3ε-ζ+表达的影响;定量ELISA法测定上清中TGF-β1、VEGF、IL-4、IL-6、IL-10和PGE2含量。结果 Colon26细胞可使5项免疫功能指标明显受抑制,抑制率分别为37.88%±7.38%、68.30%±10.44%、69.60%±5.15%、68.15%±2.17%、23.34%±2.86%。Colon26细胞可稳定分泌免疫抑制分子,以TGF-β1含量最高,为(1295.23±29.85)pg/ml;VEGF、IL-4、IL-6、IL-10、PGE2分泌量分别为(29.18±1.90)pg/ml、(27.70±0.70)pg/ml、(26.55±0.60)pg/ml、(29.52±2.50)pg/ml、(9.69±0.42)pg/ml。结论 Colon26细胞对T细胞增殖、活化及信号转导水平的抑制接近70%,对NK杀伤及ζ链介导的活化信号水平的抑制约30%。结直肠癌细胞可能通过分泌以TGF-β1为主的多种免疫抑制分子抑制NK细胞和T细胞免疫功能,介导肿瘤的免疫逃逸。

Objective To analyze the patterns of immunosuppression and immunosuppressor secretion in colorectal tumor cells.Methods Cultured supernatants of Colon26 （murine colorectal tumor cells） were collected. The transformation of T lymphocyte and NK killing activity were measured by MTT; the IL-2Rα, CD3ε＋ζ＋ and CD3ε-ζ＋ expressions were detected by FCM of direct immunofluorescence labeling. The concentrations of six immunosuppressors （including TGF-β1, VEGF, IL-4, IL-6, IL-10 and PGE2） in supernatants were determined by quantitative ELISA.Results Supernatant of Colon26 inhibited all of the five immunofunctions significantly, with inhibitory rates 37.88%±7.38%, 68.30%±10.44%, 69.60%±5.15%, 68.15%±2.17% and 23.34%±2.86% respectively.Immunosuppressors were secreted steadily in the supernatant of Colon26; the concentration of TGF-β1 was the highest, with a value about （1295.23±29.85） pg/ml; VEGF, IL-4, IL-6, IL-10 and PGE2 were（29.18±1.90）pg/ml,（27.70±0.70 ）pg/ml,（26.55±0.60）pg/ml,（29.52±2.50）pg/ml and（9.69±0.42）pg/ml respectively.Conclusion 70% of proliferation, activation and signal transduction of T cells, and about 30% of killing activity and signal transduction mediated by ζ chain of NK cells were inhibited by Colon26.Murine colorectal tumor cells impeded the innate and adaptive immune surveillance of NK and T cells through secreting immunosuppressors mainly consisting of TGF-β1 to escape anti-tumor immunity.