自体树突状细胞活化的骨髓细胞对慢性粒细胞白血病细胞毒性作用研究

Autologous dendritic cells eliciting cytotoxicity of bone marrow cells against chronic granulocytic leukemia

目的 考察自体树突状细胞 (DC)活化的骨髓细胞对慢性粒细胞白血病 (CGL)细胞的毒性作用。方法 用免疫磁珠从 2例缓解的CGL患者骨髓单个核细胞 (BMMNC)分离DC ,另取BMMNC分成 3份 ,分别加入长期培养基 ,建立Dexter培养体系。第 1份为对照 ,第 2份加重组人白细胞介素 2(rhIL 2 ) ,第 3份于培养第 4天加入收获的DC ,培养 10d后收集非贴壁细胞。以上述 3份非贴壁细胞为效应细胞 ,对照非贴壁细胞为靶细胞 ,采用双色流式细胞技术进行细胞毒性实验 ,同时用流式细胞技术检测BMMNC的P2 10表达。结果 DC活化的效应细胞比rhIL 2活化的效应细胞对靶细胞有更大的杀伤作用 ,靶细胞中的死细胞比例例 1分别为 6 3.12 %和 42 .5 9% ,例 2分别为 6 1.6 0 %和 2 1 46 %。另外 ,DC活化的效应细胞与靶细胞混合孵育后 ,效应细胞本身的死细胞比例明显增加 ,而rhIL 2活化的效应细胞无此现象。BMMNC经培养后非贴壁细胞中P2 10阳性细胞比例明显减少 ,以含DC者最为显著 ,含rhIL 2者次之。结论 自体DC活化的骨髓细胞能对CGL细胞产生明显的细胞毒性作用 ,DC的作用胜过rhIL 2。这些经DC活化的骨髓回输后可能在体内发挥移植物抗白血病 (GVL)效应。

Objective To investigate the activity of bone marrow cells activated by autologous dendritic cells(DC) to mediate cytotoxicity against chronic granulocytic leukemia(CGL) cells. Methods DC were separated from bone marrow mononuclear cells(BMMNC) of two CGL patients in hematological remission and harvested after 3 days of culture in IMDM containing autologous plasma, rhGM CSF and rhTNFα at 37 ℃, 5% CO 2 humidified atmosphere. BMMNC obtained from the patients were divided into 3 groups to set up Dexter systems: the control group, rhIL 2 containing, and the third group having DC added at day 4. After 10 days of culture, non adherent cells were harvested and the changes of immunological phenotype and the percentage of P210 positive cells were analyzed. The cytotoxicity were assayed with two colour flow cytometry. The non adherent cells from all the 3 systems served as effector cells, those from control system as target cells. Results The cytotoxic activity against target cells was greater in the DC activated effector cells than that in rhIL 2 activated ones. The percentages of death cells in target cells were 63.12% versus 42.59%(case 1) and 61.60% versus 21.46% (case 2), respectively. In addition, there was a marked increase in the death cell percentage in the DC activated effector cells themselves after incubation with target cells. This phenomenon was not found in the rhIL 2 activated effector cells. The percentage of P210 positive cells was significantly lower in non adherent cells after 10 days of culture in Dexter system, comparing with that in non cultured BMMNC. The least P210 positive cells were found in those cultured with DC and the less in those with rhIL 2. Conclusion Autologous DC were able to activate bone marrow cells to generate cytotoxicity against CGL cells. Their effect was greater than that of rhIL 2. These activated bone marrow cells might mediate graft versus leukemia effect in vivo.