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为研究CD40配体(CD40L)刺激的负载肿瘤抗原的树突状细胞(DC)活化的特异性细胞毒性T淋巴细胞(CTL)对结肠癌细胞的体外杀伤作用。应用粒细胞-巨噬细胞集落刺激因子(rhGM-CSF)、白细胞介素4(IL-4)和肿瘤坏死因子α(TNF-α)诱导培养外周血DC,并对其形态、表型及功能进行鉴定。冻融法获取结肠癌细胞抗原并联合rshCD40L刺激DC,激活自体T淋巴细胞,制备特异性CTL,MTT法检测对结肠癌细胞的体外杀伤作用。ELISA法检测γ干扰素(IFNγ-)分泌情况。结果显示,外周血单个核细胞(PBMC)经细胞因子诱导后具有典型的DC形态,高表达MHCⅡ类分子(67.70%±6.22%)、CD80(63.32%±5.49%)、CD86(70.61%±6.61%)、CD40(44.30%±1.35%)、CD1a(57.65%±3.23%),具有极强的激发MLR的能力。CD40L刺激的负载结肠癌抗原DC激活的CTL表现出对结肠癌细胞的特异性杀伤作用,产生高水平的IFN-γ,且与未经CD40L刺激的DC疫苗相比有显著性差异(P<0.05);未负载结肠癌抗原DC刺激的CTL对结肠癌细胞无杀伤作用。结果表明,应用细胞因子从人外周血中诱导出的DC负载结肠癌抗原后,激活的CTL在体外对结肠癌细胞能产生特异性杀伤作用,联合CD40L能增强其CTL的杀伤作用。
To investigate the in vitro killing effect of CD40 ligand (CD40L) on tumor cells loaded with tumor antigen (DC) activated specific cytotoxic T lymphocytes (CTLs) on colon cancer cells. Peripheral blood DCs were induced by rhGM-CSF, IL-4 and TNF-α, and their morphology, phenotype and function Identification. Freeze-thaw method to obtain colon cancer cell antigen combined with rshCD40L stimulated DC, activated autologous T lymphocytes, preparation of specific CTL, MTT assay of colon cancer cells in vitro cytotoxicity. ELISA method to detect the secretion of IFN-γ. The results showed that peripheral blood mononuclear cells (PBMC) induced by cytokines had a typical DC morphology, high expression of MHC class II molecules (67.70% ± 6.22%), CD80 (63.32% ± 5.49%), CD86 (70.61% ± 6.61 %), CD40 (44.30% ± 1.35%) and CD1a (57.65% ± 3.23%). CTLs stimulated with CD40L-stimulated colon cancer antigen DCs showed specific cytotoxicity against colon cancer cells, producing high levels of IFN- [gamma] and a significant difference from DC vaccines not stimulated with CD40L (P <0.05 ); CTLs not stimulated with DC stimulated by colon cancer antigen had no killing effect on colon cancer cells. The results showed that CTL induced by cytokine inducing DC from human peripheral blood could produce specific killing effect on colon cancer cells in vitro, and CD40L could enhance CTL killing effect.