融合快速培养的树突细胞体外诱导特异性抗肝癌免疫反应

Inducing hepatocellular carcinoma-specific cytotoxic T lymphocyte response using formed by fusion of FastDCs and allogeneic human hepatocellular carcinoma cells

目的用培养健康志愿者外周血CD14+单核细胞获得的成熟树突细胞与肝癌细胞系HCCLM3细胞构建融合细胞,在体外观察和评价这类融合细胞的功能。方法从HLA-A2阳性的健康人外周血中分离出CD14+细胞,在树突细胞完全培养基中经过48h培养及促成熟获得成熟的树突细胞(称之为FastDC)。以50%聚乙二醇和10%二甲基亚砜为融合剂融合树突细胞与肝癌细胞系HCCLM3细胞,细胞融合成功后进行融合细胞刺激自体T细胞增殖及CD8+T细胞特异性杀伤实验,并与树突细胞组、HCCLM3组及树突细胞和HCCLM3混合组进行比较。结果用48h培养获得的树突细胞与肝癌细胞系HCCLM3细胞所构建的融合细胞能有效刺激自体T细胞的增殖反应,所活化的CD8+T细胞分泌IFN-γ的水平(3d为400pg/ml±60pg/ml,5d为1030pg/ml±160pg/ml,7d为1260pg/L±180pg/L)高于其他组,而且能以MHC-Ⅰ类分子途径特异性杀伤HCCLM3细胞。结论从外周血CD14+细胞48h培养所获树突细胞与肝癌细胞系HCCLM3细胞构建的融合细胞体外能有效诱导特异性杀伤HCCLM3细胞的免疫反应。

Objective To fuse human hepatocellular carcinoma （HCC） cells with mature monocyte-derived dendritic cells （FastDC） and to observe in vitro the function of the fused cells in stimulating autologous T cells proliferation and inducing HCC-specific cytotoxic T lymphocyte （CTL） response. Methods CD14^＋ cells were isolated and purified from the peripheral blood of a healthy HLA-A2 blood donor and cultured in fresh dendritic cell complete medium for 24 h, then proinflammatory mediators were supplemented for another 24 h, thus generating mature dendritic cell （FastDCs）. The FastDCs were fused with human HCC cells of the line HCCLM3 to generate novel dendritoma. T cells were isolated from selected CD14^- cells and then divided into 4 groups to be stimulated with dendritoma cells, DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells respectively for 96 hours. 18 hours before the end of cultivation ^3H- TdR was added into the culture fluid. Scintillation counter was used to measure the cpm values. CD8^＋T cells were isolated from CD14^- cells, and added with different stimulating cells radiated by ^60Co and IL-2, IL-6, and IL-7. The values of IFN-γ, in the supernatants of the culture fluid of CD8^＋ T cells with dendritoma cells, DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells was measured. HCCLM3, K562, HLE, serf monocytes labeled with Na2^51CrO4 were added with effector cells, γ-scintillation counter was used to measure the cpm value so as to calculate the killing ability of CTL. Results The CTLs activated by dendritoma cells specifically killed the HCCLM3 cells in the context of MHC class I and acted less vigorously against the control target cells. The CTLs activated by dendritoma cells were stronger in killing HCCLM3 cells than DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells （ all P 〈 0. 05 ）. The killing activity was decreased on the HCCLM3 cells incubated with anti-HLA-ABC antibody. Three, five, and seven days after co-cultivation the value of IFN-γ in the supernatants of the culture fluid of CD8^＋ T cells with fused cells, DCs, HCCLM3 cells, and mixed DCs-HCCLM3 cells increased gradually, especially in the supernatants of the culture fluid of CD8^＋T cells with dendritoma cells （400 pg/ml ±60 pg/ml 3 days after ,1030 pg/ml ±160 pg/ml 5 days after, and 1260 pg/L ±180 pg/L 7 days after）. Condusion The novel dendritomas formed with HCCLM3 cells and mature FastDCs from healthy human peripheral blood CD14 ^＋ monocytes are potent stimulators for CD8^＋T cells in inducing HCCLM3 cell-speciflc lysls. With shorter time required for in vitro DC development, the rapid method of generation of dendritoma is more economic and may represent a new strategy for immunotherapy of hepatocellular carcinoma.