【作者】 杨昆；

【导师】 李强；

【作者基本信息】 四川大学 ， 儿科学， 2007， 博士

【摘要】 背景和目的儿童急性髓性白血病（AML）治疗效果差，容易复发，复发的根源为体内微小残留病（MRD）的存在，而MRD的根除需要有效启动T细胞介导的细胞毒免疫反应。然而，AML白血病细胞MHC、共刺激分子和黏附分子表达量低或不表达，不能将白血病抗原有效地提呈给T细胞从而启动机体特异性抗白血病免疫应答。因此，清除MRD而达到防止复发和根治白血病的关键是将白血病抗原有效地提呈给T细胞并激活T细胞介导的抗白血病免疫应答。树突状细胞（DC）是目前功能最强，也是唯一可直接活化初始型T细胞的抗原提呈细胞，其独特的功能在肿瘤免疫中备受关注。用细胞因子（CK）体外诱导培养DC的方法现己非常成熟，但存在代价昂贵、培养时间长、易污染以及某些急性髓性白血病不能诱导生成DC等缺点。近年发现钙动员可快速诱导外周血单个核细胞、CD34^＋造血祖细胞等正常细胞分化为DC。本实验第一部分用钙离子载体A23187将对多种细胞因子诱导不敏感的髓系白血病细胞株HL-60细胞快速诱导分化为成熟DC，并对其形态学、免疫表型及刺激T淋巴细胞增殖功能进行分析；第二部分引入蛋白激酶C（PKC）特异性抑制剂Bis-1，初步探讨细胞内PKC信号途径在A23187诱导HL-60细胞向DC分化过程中的作用，了解细胞内钙信号转导途径与PKC信号途径是否存在相互应答；第三部分探讨临床AML患儿不同FAB亚型的原代白血病细胞在体外被钙离子载体A23187诱导生成DC的可能性。白血病来源的DC不仅携带着白血病细胞的全部抗原信息，而且不会发生排斥反应，为临床彻底清除AML患者体内的微小残留病提供了一条前景乐观的途径。方法1.用钙离子载体A23187或联合rhIFN-（?）诱导髓系白血病细胞株HL-60细胞分化为DC，用倒置显微镜和扫描电镜观察其形态学特征，流式细胞仪检测其免疫表型，混合淋巴细胞反应检测其刺激T淋巴细胞的能力。2.用蛋白激酶C特异性抑制剂Bis-1预先处理HL-60细胞再用A23187处理，比较先经Bis-1处理的HL-60细胞与单用A23187处理的HL-60细胞在形态学、免疫表型及刺激T细胞增殖能力方面的异同。3.分离12例初诊急性髓性白血病患儿的骨髓或外周血单个核细胞，用A23187培养3～4天，进行形态学、免疫表型及功能的检测和分析。3.用PKC特异性抑制剂Bis-1预先处理再予A23187处理的HL-60细胞其形态、免疫表型及刺激T淋巴细胞增殖的能力均受到不同程度的抑制，细胞表面DC特异抗原CD83、CDS0、CD86分子的阳性表达率分别为（13.23±2.15）％、（9.70±1.69）％、（23.37±7.50）％，与单用A23187处理的HL-60细胞比较，有统计学差异（P值均＜0.05）。4.12例AML患者中有10例患者（其中M₁型1例、M₂型1例、M₃型2例、M₄型1例、M₅型5例）的白血病细胞经A23187培养3～4d后出现符合典型DC形态的细胞，但有2例AML患者白血病细胞的形态始终无明显变化；12例AML患者中有10例患者的白血病细胞经A23187诱导后出现CD83、CD80、CD86分子表达升高：培养前细胞CD83、CD80、CD86分子的阳性表达率分别为（2.06±0.95）％、（2.24±1.05）％、（3.61±1.43）％，经A23187诱导后，CD83、CD80、CD86分子表达均明显增高，分别为（28.19±8.21）％、（45.32±9.90）％、（58.67±12.17）％，与培养前相比有统计学差异（P值均＜0.05）。12例AML患者中有10例患者的白血病细胞经A23187诱导后刺激T淋巴细胞增殖的能力明显增强。此外，AML患儿M₄／M₅型与非M₄／M₅型来源的AML细胞在A23187诱导前后免疫表型比较发现，M₄／M₅型的白血病细胞更易诱导生成DC样细胞，其细胞表面成熟DC免疫标记CD83、共刺激分子CD80、CD86的表达率均明显高于非M₄／M₅来源的DC，差异有统计学意义（P值＜0.05）。外周血与骨髓来源的白血病细胞诱生为DC的表面免疫标志的表达率的差异无统计学意义（P值＞0.05）。结论1.钙离子载体A23187可使急性髓细胞白血病细胞株HL-60细胞快速诱导分化为成熟DC。2.单用IFN-（?）处理HL-60细胞，未能获得DC样细胞；联合CI-A23187诱导HL-60细胞可获得更成熟、刺激T细胞增殖能力更强的DC。3.PKC特异性抑制剂Bis-1在A23187诱导HL-60细胞分化为DC的过程中起抑制作用，表明PKC信号路径参与A23187诱导AML白血病细胞分化为DC的过程。4.多数AML患者的白血病细胞经A23187诱导3～4d分化为DC，部分病例不能诱导成DC，表明AML因具有生物学异质性而对同一种诱导剂的反应不同。M₄／M₅型白血病细胞体外培养易生成具有树突状细胞形态、特征的细胞。AML患儿外周血和骨髓样本来源的DC表面标志表达率无统计学差异，对于外周血白血病细胞比率高的病人，可采集外周血样本来取代骨髓样本诱导白血病DC。更多还原

【Abstract】 ObjectiveThe prognosis of childhood acute myeloid leukemia (AML) is very poor because the minimal residual disease (MRD) always leads to relapse. In order to eliminate MRD, it is necessary to activate antigen specific cytotoxic T lymphocytes to start anti-leukemia cytotoxic response. However, AML cells may not present specific leukemic antigen to T lymphocytes because of low expression or absence of MHC molecules, co-stimulatory and adhesion molecules. Dendritic cells (DCs) are most potent antigen-presenting cells (APCs) that prime naive T lymphocytes and activate antigen specific cytotoxic T lymphocytes. The superior ability of DCs to present antigens to T cells has led to the development of DC-based strategies of enhancing the T-cell mediated cytotoxic killing against tumors. It is well known that DC-like cells can be obtained with conventional cytokine combinations in vitro. But the methods are expensive, time consuming, easily contamination, and sometimes AML cells can not be induced into DCs. Recent years, there were reports that normal monocytes(Mos) and CD34~+ hematopoietic progenitor cells (HPCs) could be induced rapidly into DCs by calcium ionophore (CI). In the studies presented here, we at first showed the morphology, immunophenotype and functions of DCs induced by CI-A23187 from HL-60 cells, and then investigated the role of protein kinase C (PKC) signal transduction in this process by using PKC inhibitor Bis-1. At last we explored the possibility of AML cells from patients with different FAB subtypes being induced into DCs by CI-A23187. The DCs derived from leukemia cells carry all auto-leukemic antigens and do not lead to autoimmune diseases. The appliment of DC vaccine to AML patients might thoroughly eliminate MRD and is very optimal in the near future.Methods1. HL-60 cells were cultured with CI-A23187 or plus rhIFN-γfor different times. The morphologic features of HL-60 cells were observed under inverted microscope and scanning electronic microscope, while the cell phenotypes of HL-60 cells treated with A23187 were determined by flow cytometry. The proliferation of allogeneic human T cells stimulated by DCs was tested by mixed lymphocyte reaction (Allo-MLR).2. HL-60 cells were pretreated with protein kinase C (PKC) inhibitor Bis-1 for 24 hours followed by cultured with A23187 for another 36 hours, and the morphology, immunophenotype and function of stimulating proliferation of allogeneic T cells were compared with the cells only treated with A23187.3. Bone marrow or peripheral blood mononuclear cells were isolated from 12 initially diagnosed AML patients and then cultured with A23187 for 3 to 4 days. DCs induced from leukemia cells were confirmed by morphology, immunophenotype and their capability of activating allogeneic T cells.Results1. After the HL-60 cells were treated with A23187 (180ng/ml) for 20 hours, the dendritic appearance of some cells was found and the expressions of CD83, a characteristic marker of mature DCs, and co-stimulating molecules CD80 and CD86 were up-regulated. When HL-60 cells were cultured with A23187 for 48 hours, the expression of CD83 began to decrease. A large number of cells with typical dendritic appearance were observed after cultured for 72 hours with A23187 and the expressions of CD80 and CD86 were up-regulated continuously. Allo-MLR revealed that DCs derived from HL-60 cells treated by A23187 were potent to stimulate the proliferation of allogeneic human T cells.2. The DCs were not derived from HL-60 cells cultured with rhIFN-γalone for 96h. When rhIFN-γcombined with A23187 were used to induced HL-60 cells, the DCs obtained expressed higher levels of CD83,CD80, CD86 molecules and were more potent to stimulate the proliferation of allogeneic T cells. However, there were no statistic differences in DC marker expression and capability of stimulating the proliferation of T cells when these DCs were compared with that induced only with A23187.3. The morphological changes, surface marker expressions and ability to stimulating the proliferation of allogeneic T cells in HL-60 derived DCs were inhibited by PKC inhibitor Bis-1.The percentage of CD83、CD80、CD86 expressing on HL-60 cells induced by A23187 decreased to (13.23±2.15) %,(9.70±1.69)%,(23.37±7.50) %, respectively (p<0.05) . These results indicated that PKC signal transduction pathway involved in the process of HL-60 cells induced into DCs by A23187.4. AML cells in 10/12 patients induced by A23187 for 3 to 4 days showed dendritic-like morphologic change, with the expression of CD83、CD80、CD86 increased from (2.06±0.95) %, (2.24±1.05) %, (3.61±1.43) % to (28.19±8.21)%,(45.32±9.90) %, (58.67±12.17) %, respectively (p<0.05). The DCs induced from AML cells also acquired potent capability of stimulating T cells proliferation. Based on immunophenotype analysis, we found that AML cells from patients with FAB subtypes of M4/M5 were induced more easily into DCs than leukemic cells with non-M4/M5 subtypes. There were no differences in expression levels of CD83、CD80、CD86 between peripheral blood and bone marrow derived leukemic DCs.Conclusion1. HL-60 cells can be rapidly induced into mature DCs with calcium ionophoreA23187. 2. The DCs were not derived from HL-60 cells cultured with rhIFN-γalone. However, when IFN-γcombined with CI-A23187 were used, DCs induced from HL-60 cells acquired were more mature and more potent to stimulate the proliferation of T cells.3. The induction of HL-60 cells into DCs by A23187 can be inhibited by PKC inhibitor Bis-1, suggesting signal transduction through PKC pathway might be involved in the process of AML cells differentiated into DCs by A23187.4. The majority of AML cells from patients with different FAB subtypes can be induced rapidly into DCs with A23187 cultured for 3 to4 days in vitro. Some cases of AML are not response to A23187 induction, indicating there is biological heterogeneity in AML patients which lead to different response to A23187. AML cells derived from patients with M4/M5 subtypes are more easily induced into DCs by A23187 than that of non-M4/M5 subtypes. Peripheral blood with high percentage of blast cells in AML patients can be used to replace bone marrow as an alternative source of leukemic DCs.更多还原