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目的探讨转染自体胃癌细胞总RNA的树突状细胞(DC)体外介导抗胃癌的免疫效应。方法制备短期培养的原代胃癌细胞。用rhGM-CSF、rhIL-4和TNF-α体外诱导胃癌患者外周血单个核细胞(PBMC)中DC的发育和成熟,并转染自体肿瘤细胞总RNA,激活自体T细胞产生CTL,用CCK-8试剂盒检测CTL的杀伤活性。应用流式细胞术及混合淋巴细胞培养技术检测DC的免疫功能状态。用ELISA法测定IL-12和INF-γ的水平。结果转染自体肿瘤细胞总RNA的成熟DC,不仅可高表达MHC-I、II类分子及CD80、CD83和CD86协同刺激分子,并可获得高效刺激自体或异体T细胞增殖的能力。转染RNA的成熟DC,分泌IL-12的水平及其刺激产生的CTL培养上清液中INF-γ的水平显著高于单纯成熟DC及未成熟DC;且CTL对自体胃癌细胞的杀伤率显著高于异体组。结论转染自体胃癌细胞总RNA的成熟DC能够体外诱导产生对自体肿瘤细胞具有高度抗原特异性杀伤活性的CTL。
Objective To investigate the immunological effects of dendritic cells (DCs) transfected with total RNA of autologous gastric cancer cells in vitro on gastric cancer. Methods Short-term cultured primary gastric cancer cells were prepared. In vitro, the development and maturation of DCs in peripheral blood mononuclear cells (PBMCs) from patients with gastric cancer were induced by rhGM-CSF, rhIL-4 and TNF-α, and transfected with total RNA from autologous tumor cells. CTLs were activated by autologous T cells, 8 kit for the detection of CTL killing activity. Application of flow cytometry and mixed lymphocyte culture technology to detect DC immune function status. The levels of IL-12 and INF-γ were measured by ELISA. Results Mature DCs transfected with total RNA of tumor cells not only expressed MHC-I, II molecules and co-stimulatory molecules of CD80, CD83 and CD86, but also acquired the ability to efficiently stimulate the proliferation of autologous or allogeneic T cells. The level of IL-12 secretion and the level of INF-γin CTL culture supernatant secreted by transfected RNA were significantly higher than those of pure DCs and immature DCs. The cytotoxicity of CTL on autologous gastric cancer cells was significantly Higher than allogeneic group. Conclusion Mature DCs transfected with total RNA from human gastric cancer cells can induce the production of CTLs with high level of antigen-specific killing activity on autologous tumor cells in vitro.