摘要
目的检测应用脐血诱导成熟树突状细胞(DC),并以急性白血病(AL)总RNA冲击该DC所诱导产生的特异性细胞毒T细胞(CTL)的杀伤活性。方法取AL患者外周血或骨髓,以淋巴细胞分离液获得单个核细胞(MNC),并提取RNA。分离脐血MNC,以2×106个细胞/孔接种于12孔培养板,培养体系中加入重组人粒单核细胞集落刺激因子(rhGM-CSF),重组人白细胞介素4(rhIL-4)和肿瘤坏死因子α(TNF-α),并在培养第5天,加入白血病总RNA,致敏脐血DC。培养过程中,观察树突状细胞形态,流式细胞仪检测表型。培养第12天收获成熟DC,与自体T淋巴细胞共培养,诱导产生白血病特异性CTL,乳酸脱氢酶法(LDH法)测定溶细胞活性。结果脐血来源DC表现出成熟DC典型形态和免疫表型特征,培养第5天,DC相关分化抗原CD1a、CD83、CD86、CD80的表达较培养前增高,差异有统计学意义(P<0.01);培养至第12天,DC相关分化抗原CD1a、CD83、CD86、CD80的表达较培养第5天增高,另外人类白细胞相关抗原(HLA-DR)的表达也较培养第5天增高,差异均具有统计学意义(P<0.01)。以白血病RNA转染的DC组所刺激的自体T淋巴细胞对自身白血病靶细胞显示出明显的杀伤活性,而未经抗原转染DC组无明显杀伤活性,按照各效靶比进行两者的比较,差异有统计学意义(P<0.01)。结论利用细胞因子体外诱导脐带血产生大量成熟DC,以急性白血病RNA转染此DC,并以此白血病特异性DC诱导产生抗白血病特异性CTL,不失为清除AL患者体内残留灶的积极措施。
Objective To explore the activity of the leukemia-specific cytolytic T lymphocyte （CTL） stimulated by the dendritic cells （DCs） from cord blood transfected with the total RNA of acute leukemia（AL） cells. Methods Peripheral blood or bone marrow mononuclear cells （MNCs） were isolated from AL patients with lymphocytes separation medium. The total RNA was extracted from these MNCs. The cord blood MNCs （2 × 10^6 cells in each well） were cultured in vitro in the presence of combined cytokines （rhGM-CSF, rhIL-4 and TNF-α） to generate the mature DCs,and were transfected with the total RNA on day 5. The morphology of the DCs was observed during the culture. After 12-days＇ culture, the cells were harvested and the immune phenotype of the DCs were identified by flow cytometer （FCM）. The auto-T lymphocytes were cultured with the DCs transfected with the total RNA. Cytotoxicity was determined by lactate dehydrogenase （LDH） release method. Results After 5-days＇ culture, the cord blood DCs developed classical morphologic and phenotypic characteristics of mature DCs. On day 5, the DC-associated surface molecules such as CD1α, CD83, CD86, CD80 were upregulated, and on day 12, these surface molecules and human leucocyte antigen DR （HLA-DR） were upregulated compared with those on day 5. The CTLs exhibited significant killing activity to auto-AL target cells compared with the control （ P 〈0.01）. Conclusion The combined cytokines could induce the cord blood cells into mature DCs in vitro. AL specific CTL responses could be generated from auto-T lymphocytes stimulated by the DCs transfected with the total RNA of AL cells in vitro. This study provides a potent method to elucidate the minimal residual disease of AL.
出处
《临床荟萃》
CAS
北大核心
2007年第2期80-83,共4页
Clinical Focus
关键词
白血病
髓样
胎血
树突细胞
T细胞
免疫
leukemia, myeloid
fetal blood
dendritic cells
T-cell
immunity
