胸腺五肽对黑色素瘤细胞诱导的肿瘤浸润树突状细胞激活作用观察

Effect of thymopentin on activation of tumor infiltrating dendritic cells induced by melanoma cells

目的观察胸腺五肽(TP5)对黑色素瘤细胞诱导的肿瘤浸润树突状细胞(TIDCs)的激活作用。方法将对数生长期的黑色素瘤细胞株B16在无血清培养基中培养24 h,收集上清即B16细胞条件培养基;从BALB/c小鼠股骨中收集细胞悬液,加入IL-4、重组鼠粒细胞/巨噬细胞集落刺激因子(GM-CSF)和50%B16细胞条件培养基诱导培养,倒置相差显微镜拍照鉴定TIDCs。分别将0、10、20、40μmol/L的TP5加入TIDCs中,记为0μmol/L组、10μmol/L组、20μmol/L组、40μmol/L组,37℃共同孵育48 h,分离出TIDCs细胞和上清液。取收集到的TIDCs细胞,加入荧光标记的CD11c、CD86及MHCⅡ抗体,用流式细胞仪检测TIDCs表面成熟标记分子CD11c、CD86、MHCⅡ。取TIDCs细胞上清液,按ELISA试剂盒说明书测定IL-2和IL-10。向TIDCs细胞培养皿中加入FITC标记的1μg/m L的TP5溶液200μL,激光共聚焦显微镜下观察TP5与TIDCs的结合情况。结果倒置相差显微镜低倍镜下可见细胞聚集成集落的细胞群,呈半贴壁状态;高倍镜下可见细胞已经呈现典型的树突状形态,有的细胞出现短的出芽状突起,有的细胞已经出现长长的突起,提示TIDCs诱导培养成功。与0μmol/L组相比,40μmol/L组TIDCs中CD11c、CD86、MHCⅡ、IL-12的表达量升高(P均<0. 05),IL-10的表达量降低(P <0. 05);与10μmol/L组和20μmol/L组相比,40μmol/L组TIDCs中CD86、IL-12的表达量升高(P均<0. 05)。激光共聚焦显微镜下可见,TIDCs的细胞核呈椭圆形,FITC标记的TP5分布在细胞核周围,提示TP5结合在TIDCs的细胞膜上及胞浆中。结论 TP5可促进TIDCs表面成熟标志分子CD11c、CD86及MHCⅡ的表达,促进TIDCs分泌IL-12及抑制IL-10的分泌,表明TP5能够激活免疫活性受抑制的TIDCs的抗原递呈功能。

Objective To investigate the activation of tumor infiltrating dendritic cells(TIDCs)by thymopentin(TP5).Methods The melanoma cell line B16 in logarithmic growth phase was cultured for 24 h in serum-free medium,and the supernatant was collected to form B16 cell conditioned medium.The cells were collected from the femur of BALB/c mice,and were induced by IL-4,recombinant mouse granulocyte/macrophage colony-stimulating factor(GM-CSF),and 50%B16 cell conditioned medium,and TIDCs were identified by inverted phase contrast microscopy.TP5(0,10,20,and 40μmol/L)were added to TIDCs,which were recorded as the 0μmol/L group,10μmol/L group,20μmol/L group,and 40μmol/L group,and were co-incubated with TIDCs at 37°C.After incubation for 48 h,TIDCs cells and the supernatant were isolated.The TIDCs cells were incubated with fluorescently labeled CD11c,CD86 and MHCII antibodies,and flow cytometry was used to detect the expression of CD11c,CD86 and MHCII in TIDCs.ELISA kit was used to detect IL-2 and IL-10 in TIDCs.FITC-labeled TP5 solution(1μg/mL,200μL)was added to TIDCs,and the binding of TP5 to TIDCs was observed using laser confocal microscope.Methods Under low magnification,the cells were semi-adherent,and under high magnification,the cells had a typical dendritic morphology,some cells had short bud-like protrusions,and some had long protrusions,suggesting that TIDCs were successfully induced.Compared with the 0μmol/L group,the expression of CD11c,CD86,MHCII and IL-12 in the 40μmol/L TP5 group increased(P<0.05),and the expression of IL-10 decreased(P<0.05).Compared with the 10μmol/L and the 20μmol/L TP5 group,the expression of CD86 and IL-12 in the 40μmol/L TP5 group increased(P<0.05).Under the confocal microscope,the nucleus of TIDCs was elliptical,and FITC-labeled TP5 was distributed around the nucleus,suggesting that TP5 bound to the cell membrane and cytoplasm of TIDCs.Conclusion TP5 can promote the expression of CD11c,CD86 and MHCII in TIDCs,promote the secretion of IL-12 and inhibit the secretion of IL-10,indicating that TP5 can activate the antigen-presenting function of TIDCs with suppressed immunological activity.