改良急性髓系白血病细胞致敏的自体DC-CIK细胞抗AML的分子靶标

DC-CIK cell is the important candidate cell in immunotherapy of malignancies. Our research group has carried out the early clinical trials of autologous DC-CIK cells in the treatment of acute myelogenous leukemia (AML). In order to enhance the efficacy of autologous DC-CIK cell immunotherapy in the treatment of AML. culture methods of autologous DC-CIK cell need to be improved, and the anti-AML molecular target need to be explored. In this project, the autologous primitive cells are re-sensitized in culturing autologous DC, the autologous primitive cells are re-sensitized in culturing autologous DC, then added with interleukin-15 with up- effect T and down-Treg cells for CIK co-culture. By this way, the autologous DC-CIK with special anti-AML effect is cultured.The surface molecules of AML cells, DC cells, and autologous DC-CIK cell will be evaluaged by cell microarray; the changes of key signal molecules are detected using the signal transduction pathway PCR chip, and the anti-AML molecular target is screened. Based on the screening results, the functional recovery verification is done for the lentivirus carried with molecular target as well as the CIK cell from lentivirus transfection by molecular target RNA intervention, and the molecular target in AML patients is verified for the DC-CIK treatment. This research project will lay foundation for development of AML special cellular immunotherapy.

DC-CIK是细胞免疫治疗恶性肿瘤中的重要候选细胞。课题组已开展急性髓系白血病（AML）细胞诱导分化的自体DC-CIK细胞治疗AML的前期临床试验。为提高自体DC-CIK细胞免疫治疗AML的疗效，需改良AML自体DC-CIK细胞培养方法，探索抗AML作用的分子靶标。本课题在培养自体DC过程中采用自体原始细胞再次致敏，与加入具有上调效应T细胞，下调抑制T细胞作用的白介素-15培养的CIK共培养， 培养具有特异性抗AML作用的自体DC-CIK细胞。应用细胞芯片检测AML细胞、DC细胞及自体DC-CIK细胞表面分子；应用信号转导通路PCR芯片检测关键信号分子的变化，并筛选抗AML分子靶标。据筛选结果，将搭载分子靶标的慢病毒和搭载针对分子靶标RNA干扰的慢病毒转染CIK细胞进行功能回复性验证，并验证DC-CIK细胞治疗AML病人体内分子靶标。本课题将为特异性细胞免疫治疗AML的发展和改进奠定基础。

背景：树突状细胞(DC)是一种特殊的抗原提呈细胞，细胞因子诱导的杀伤细胞对多种肿瘤具有特异性杀伤活性。最近的研究证实，共培养的DC-CIK细胞具有高效的抗肿瘤活性，是肿瘤生物免疫治疗的重点。然而，DC-CIK细胞在急性髓系白血病(AML)中的作用机制和功能仍然很难确定。.材料和方法：通过高通量测序，在健康和AML患者的DC-CIK细胞中获得转录物。通过RT-qPCR法验证了大差异表达的mRNA，选择MMP9和CCL1进行后续的体内和体外实验研究。.结果：MMP9和CCL1在AML患者DC-CIK细胞中高表达。单独用MMP9和CCL1敲除的DC-CIK对AML细胞影响不大，而在DC-CIK细胞中敲除MMP9和CCL1可降低细胞毒性，抑制AML细胞的增殖和诱导细胞凋亡，AML细胞被T细胞激活。此外，我们还证明MMP9和CCL1沉默的DC-CIK显著提高了CD3+CD4+和CD3+CD8+细胞并减少了CD4+PD-1+,CD8+PD-1+T细胞。同时，DC-CIK中MMP9和CCL1的阻断显著增加了IL-2和IFN-γ，增加了CD107aþ(LAMP1)和颗粒酶B(GZMB)，下调了AML患者和AML模型小鼠的PD-1、CTLA-4、TIM-3和LAG-3T细胞。此外，在DC-CIK中的MMP9和CCL1敲除也阻止了AML细胞与活化的T细胞共培养后的增殖和加速凋亡。.结论：我们的研究结果表明，阻断DC-CIK细胞中MMP9和CCL1可通过激活T细胞显著提高AML的治疗效率。