树突细胞应用于荷白血病小鼠免疫治疗的实验研究

目的探讨白血病抗原活化的树突细胞(DC)应用于荷白血病小鼠异基因骨髓移植 (allo-BMT)后免疫治疗的可行性及有效性。方法应用mGM-CSF及mIl-4从小鼠骨髓细胞扩增出成熟DC,使其负载经冻融法制备的L7212白血病细胞相关抗原。将进行allo-BMT后的荷白血病小鼠分 A(对照组)、B(T细胞组)、C(DC T细胞组)三组进行免疫治疗,观察小鼠生存率、生存期、移植物抗宿主病(GVHD)发生等情况及血清细胞因子水平和脾细胞细胞毒活力变化。对各组长期生存小鼠行二次攻击,以观察其体内抗白血病免疫能力。结果小鼠骨髓单个核细胞经mGM-CSF、mIL-4联合作用7 d可生成大量成熟DC。B、C二组复发率分别为30％及0％,移植后长期生存率分别为30％及 70％,两组差异均有统计学意义(P<0．05),而GVHD发生率差异无统计学意义。两组平均生存时间分别为(32．95±13．29)d、(41．15±13．88)d,差异有统计学意义(P<0．01)。MYT法检测发现C组小鼠脾细胞对L7212细胞产生特异性杀伤作用;EIJSA法检测显示血清中Th1类因子IL-2水平为 (419．75±26．66)pg／ml,明显高于其他两组(P<0．01)。二次攻击后B、C二组生存率分别为33．3％及85．7％,差异有统计学意义(P<0．05)。结论肿瘤抗原负载的DC联合供者淋巴细胞输注是增强 allo-BMT后移植物抗白血病效应、减少白血病复发的有效手段。

Experimental study on immunotherapy with dendritic cell in leukemic mice model

OBJECTIVE: To explore the feasibility and efficiency of immunotherapy with dendritic cell (DC) in leukemic mice model after allogeneic bone marrow transplantation (allo-BMT). METHODS: Mature DC were expanded from mice bone marrow mononuclear cells (MNC) by adding mouse granulocyte-macrophage colony stimulating factor (mGM-CSF) and interleukin-4 (mIL-4). Three days later they were pulsed with frozen thawing L7212 leukemia-related antigen. Mice bearing leukemia received allo-BMT at d 0, and then were divided into control group (A), T cells group (B) and DC + T cells group (C) to receive respective immune therapy at d 14. The survival rate, survival time, occurrence of graft-versus-host disease (GVHD), cytotoxicity of spleen cells and serum cytokine level were observed. The survivors in each group were rechallenged with L7212 cells to observe the immune response to the leukemia. RESULTS: Mature DC were successfully induced from bone marrow MNC. In groups B and C, the relapse rates were 30% and 0%, while the long term survival rates after BMT was 30% and 70% respectively. Both of the differences were statistically significant (P < 0.05). However, the incidence of GVHD in these two groups were similar. The mean survival times were (32.95 +/- 13.29) days and (41.15 +/- 13.88) days, respectively (P < 0.01). MTT assay indicated that spleen cells from group C had specific killing activity to L7212 cells. Enzyme-labeled immunosorbent assay (ELISA) showed that the serum IL-2 level in group C was (419.75 +/- 26.66) pg/ml, being significantly higher than that in the other two groups (P < 0.01). When the survivors were rechallenged with L7212 cells, there was difference between the survival rates of groups C and B (85.7% vs 33.3%, P < 0.05). CONCLUSION: Immunotherapy with leukemia related antigen-pulsed DC in combination with donor lymphocyte infusions is an effective approach to reinforce GVL effect and decrease relapse after allo-BMT.