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目的探讨负载自体肿瘤细胞裂解物的成熟树突状细胞(ATLs-mDCs)体外诱导个体化抗胃癌过继免疫治疗的效应。方法建立短期培养的原代胃癌细胞系。用ATLs-mDCs激活自体T细胞,制备肿瘤特异性细胞毒性T细胞(CTLs)。自体树突状细胞(DCs)均分为未成熟DCs、成熟DCs和ATLs-mDCs 3组,分别应用流式细胞仪及混合淋巴细胞增殖反应方法,检测DCs的免疫功能状态;应用细胞毒杀伤试验验证肿瘤特异性CTLs的杀伤活性;应用酶联免疫吸附试验(ELISA)测定DCs培养上清中白细胞介素12(IL-12)和CTLs上清中γ干扰素(IFN-γ)的分泌水平。结果ATLs-mDCs上调HLA-DR、CD80、CD83和CD86的表达水平,同时获得高效刺激自体T细胞增殖的能力。ATLs-mDCs诱导产生的CTLs对自体胃癌细胞的杀伤率为(84±11)%,显著高于对两株异体胃癌细胞的杀伤率[(19±7)%和(19±11)%(t=54.18和56.46,P值均<0.01)]。ATLs-mDCs上清液中IL-12的浓度显著高于单纯成熟DCs(t=15.47,P<0.01)及未成熟DCs(t=28.44,P<0.01)。3组DCs分别激活自体T细胞产生的CTLs上清液中INF-γ的浓度ATLs-mDCs组高于单纯成熟DCs组(t=4.84,P<0.05),并显著高于未成熟DCs组(t=13.74,P<0.01)。结论ATLs-mDCs在体外诱导产生的CTLs能有效特异性杀伤自体胃癌细胞。
Objective To investigate the effect of adoptive immunotherapy against gastric cancer in vitro induced by mature dendritic cells (ATLs-mDCs) loaded with autologous tumor cell lysate. Methods Short-term culture of primary gastric cancer cell lines was established. Autologous T cells were activated with ATLs-mDCs to produce tumor-specific cytotoxic T-cells (CTLs). Autologous dendritic cells (DCs) were divided into three groups: immature DCs, mature DCs and ATLs-mDCs. Flow cytometry and mixed lymphocyte proliferation assay were used to detect the immune function of DCs. Cytotoxicity assay The cytotoxic activity of tumor-specific CTLs was assayed. The levels of interleukin-12 (IL-12) and IFN-γ in the supernatants of CTLs in DCs were detected by enzyme linked immunosorbent assay (ELISA). Results ATLs-mDCs up-regulated the expression of HLA-DR, CD80, CD83 and CD86, and at the same time acquired the ability to efficiently stimulate autologous T cell proliferation. The killing rate of CTLs induced by ATLs-mDCs on autologous gastric cancer cells was (84 ± 11)%, which was significantly higher than that of CTLs (19 ± 7)% and (19 ± 11)% (t = 54.18 and 56.46, both P <0.01)]. The concentration of IL-12 in ATLs-mDCs supernatant was significantly higher than that of pure DCs (t = 15.47, P <0.01) and immature DCs (t = 28.44, P <0.01). The concentration of INF-γin CTLs supernatant produced by autologous T cells activated by 3 groups of DCs was higher than that of mature DCs group (t = 4.84, P <0.05), and significantly higher than that of immature DCs group = 13.74, P <0.01). Conclusion CTLs induced by ATLs-mDCs in vitro can effectively kill autogenous gastric cancer cells.