体外培养DC-CIK细胞杀伤白血病细胞的免疫效应机制研究

Research on immune effects and mechanisms from culturing DC-CIK in vitro and killing leukemic cells

目的研究体外培养杀伤细胞(Cytokine-induced killer cell,DC-CIK)杀伤白血病细胞的效应机制,为研究白血病的免疫疗法提供一定的试验依据。方法收集健康成年人外周血,通过淋巴细胞分离液分离获得人单个核细胞(PBMNC),经过诱导培养获得DC和CIK,通过流式细胞仪检测DC和CIK的细胞表面抗原,共培养成DC-CIK细胞。MTT法绘制DC-CIK细胞生长曲线。K562/A02,THP-1及HL-60细胞作为靶细胞,分别以DC、CIK、DC-CIK细胞作为效应细胞,并通过MTT比色法检测细胞的杀伤效应。用ELISA分别检测DC、CIK、DC-CIK细胞上清中,IL-12、IL-17、INF-α及IFN-γ的表达量,并检测细胞表面抗原。进一步通过Real-time PCR分别分析K562/A02及与DCCIK共培养后K562/A02细胞中多药耐药基因1(MDR1)的表达差异。结果从外周血中可以分离获得较纯的DC及CIK细胞,通过共培养获得DC-CIK细胞。K562/A02,THP-1及HL-60细胞作为靶细胞,分别以DC、CIK、DC-CIK细胞作为效应细胞,随着效靶比的升高,杀伤活性逐渐上升,且DC-CIK组的杀伤活性在同效靶比组中最强(P<0.01)。当K562/A02与DC-CIK细胞共培养后,其MDR1基因的表达量显著降低(P<0.01)。结论体外培养DCCIK细胞可以显著提高抗K562/A02细胞活性,主要作用机理包括显著增强IL-17等免疫因子的表达以及降低MDR1的表达。

Objective To investigate the mechanism of leukemia therapy by dendritic cells-induced killer cells（ DCCIK） through the co-cultivation of dendritic cells（ DC） and cytokine-induced killer cells（ CIK）. Methods DC and CIK cells were generated from culturing peripheral blood mononuclear cells（ PBMC） and identified by flow cytometry analysis.The cell growth curve of DC-CIK was made by MTT. The lethal effect of K562 / A02 caused by DC,CIK and DC-CIK was analyzed by MTT. In order to investigate the immune effect of leukemia therapy by DC-CIK,ELISA was used to detect IL-12,IL-17,INF-α and IFN-γ. At last,real-time PCR was used to analyze the expression of MDR1. Results The DC and CIK were successfully obtained from PBMC. Through co-cultivation,DC-CIK cell was achieved. Using K562 / A02,THP-1 and HL-60 cells as target cells,and DC,CIK,DC-CIK as effector cells,as the ratio of target cells increased,the killing activity of DC-CIK was also elevated. Among the effector cells,DC-CIK＇s lethal effect was the highest（ P 0. 01）. The expression of MDR1 was reduced in the co-cultivation of DC and CIK（ P 0. 01）. Conclusion Co-cultivation of DC-CIK cells from PBMC can significantly increase the activity of anti-K562 / A02,with its primary effect to induce IL-17 expression and reduce the expression of MDR1.