Establishment of dendritic cell immunotherapy based on the chemokine expression profile of cancer cells

已有研究表明，DC与肿瘤细胞的融合细胞可作为含有肿瘤特异性抗原的抗原提呈细胞发挥作用。几项DC/肿瘤融合细胞的临床试验表明，在疾病晚期患者中，肿瘤有明显的消退。在我们的研究中，DC与照射后的肿瘤细胞以1：1的比例混合，在含有50%聚乙二醇组分的无血清RPMI1640培养液中孵育1min以诱导细胞融合。然后，我们使用电融合技术来生产融合细胞。在初步研究中，确定了最佳排列电压(100V，10s)和最佳细胞数(1.0×10~(-6)DC：1.0×10~(-6)肿瘤细胞/毫升)。使用ECM2001电融合装置进行细胞融合，并通过荧光显微镜和流式细胞仪进行确认。树突状细胞成功地与同种异体胃癌细胞融合，产生了具有抗原提呈细胞功能的杂交细胞，因为它们诱导了同种异体CD4+T细胞的增殖。胃癌细胞株MKN-45和DC杂交瘤细胞可诱导自体的CD4+和CD8+T细胞增殖，提示MKN-45-DC杂交瘤细胞具有从MKN-45细胞到CD4+和CD8+T细胞的某些抗原。经MKN-45-DC杂交瘤细胞刺激的CD8+T细胞免疫后可杀伤MKN-45细胞。CTL杀死了另一个胃癌细胞系MKN-1和黑色素瘤细胞系888-1，这表明它们识别了一个共同的肿瘤抗原。MKN-45特异性CTL能识别癌胚抗原(CEA)，表明肿瘤抗原和同种异体抗原对CEA均有杀伤作用。DC/肿瘤融合细胞在癌症治疗中的实际应用还需要进一步的研究。进一步阐明肿瘤细胞对免疫细胞的反击机制，为肿瘤的DC治疗奠定基础。

Some previous reports indicated that fusion cells of DCs and tumor cells could function as antigen-presenting cells(APCs) containing tumor specific antigens. Several clinical trials with DC/Tumor fusion cells demonstrated an obvious tumor regression in patients with advanced stage of disease. In our study, the DCs and irradiated tumor cells were mixed at a ratio of 1:1 and incubated in serum-free RPMI 1640 medium containing 50% polyethylene glycol(PEG) for 1 min to induce cell fusion. Then, we used electrofusion techniques for producing the fusion cells. In a preliminary study, the optimal cell numbers (1.0x10^6 of DCs : 1.0x 10^6 of tumor cells/mL) and alignment voltage (100V for 10sec) were determined. Cell fusion was performed using ECM2001 electrofusion apparatus and confirmed by fluorescent microscopy and flow cytometry. The DCs were successfully fused with the allogeneic gastric cancer cells resulting in hybrid cells that functioned as antigen-presenting cells, because they induced allogeneic CD4+ T cell proliferation. A gastric cancer cell line, MKN-45, and DC hybrid cell induced autologous CD4+ and CD8+ T cell proliferation, indicating that MKN-45-DC hybrids present some antigens from the MKN-45 cells to the CD4+ and CD8+ T cells. CD8+ T cells stimulated by the MKN-45-DC hybrid cells were able to kill MKN-45 when used for immunization. The CTLs killed another gastric cancer cell line, MKN-1, as well as a melanoma cell line, 888 mel, suggesting the recognition of a shared tumor antigen. MKN-45 specific CTLs can recognize carcinoembryonic antigen(CEA), indicating that the killing is due to tumor antigens as well as alloantigens. Further studies are requested for practical use of DC/tumor fusion cells in cancer therapy. Moreover, the mechanisms of counterattack by tumor cells against immune cells should be clarified to establish an effective DC treatment for cancer.