人外周血单核细胞分化为树突状细胞过程中核转录因子-κBp50的表达及柴胡皂苷a的影响  

THE EXPRESSION OF NF-κB p50 AND THE EFFECT OF SAIKOSIDE a IN THE PROCESS OF HUMAN PERIPHERAL BLOOD MONOCYTE DIFFERENTIATING INTO DENDRITIC CELLS

作　　者：张丽[1] 唐军民[1] 唐岩[1] 李枫[1] 苏安英[2] 

机构地区：[1]北京大学基础医学院组织学与胚胎学教研室,北京100083 [2]河北工程大学临床医学院,邯郸056029

出　　处：《解剖学报》2007年第6期681-686,共6页Acta Anatomica Sinica

基　　金：国家自然科学基金资助项目(39970380)

摘　　要：目的观察人外周血单核细胞分化为树突状细胞(DC)过程中,核转录因子-κB p50(NF-κB p50)的表达以及柴胡皂苷a对单核细胞分化为树突状细胞的影响。方法非连续密度梯度离心获取人外周血单核细胞;用含粒单细胞刺激因子(GM-CSF)、IL-4、肿瘤坏死因子(TNF-α)和柴胡皂苷a的RPMI-1640培养液分别对细胞因子组、柴胡皂苷组、联合培养组的单核细胞进行体外培养;应用倒置相差显微镜、瑞氏染色和CD14、CD83、HLA-DR、S-100的免疫细胞化学法鉴定单核细胞和DC;动态观察单核细胞分化为DC过程中不同时间点的NF-κB p50的表达,并进行图像分析。结果CD14、CD83、HLA-DR、S-100免疫细胞化学法证实所获单核细胞和DC符合各自的形态和表型特征。在含GM-CSF和IL-4的RPMI-1640培养液培养7d时,单核细胞分化成未成熟DC(imDC);在含GM-CSF、IL-4和TNF-a的RPMI-1640培养液继续培养3d时,imDC分化为成熟DC(mDC),两者的细胞核内NF-κB p50表达具有显著性差异(P<0.05)。单纯用只含柴胡皂苷a的RPMI-1640培养液培养单核细胞至7d时,可见其细胞核呈NF-κBp50阳性,但未见其分化为DC。若用含GM-CSF、IL-4和柴胡皂苷a的RPMI-1640培养液联合培养7d时,则该单核细胞分化为imDC;加入TNF-α后继续培养3d,imDC的细胞核呈NF-κB p50强阳性,免疫细胞化学法证实imDC已经分化为mDC。联合培养组细胞的表型表达均强于只加细胞因子组(P<0.05)。结论人外周血单核细胞分化为imDC和mDC过程中,细胞核内NF-κB p50表达逐渐增强。单纯用低浓度柴胡皂苷a不能刺激单核细胞分化为DC,但联合细胞因子GM-CSF、IL-4和TNF-α培养时,可使其分化为imDC和mDC。Objective To observe the expression of NF-κB p50 protein in the process of the human peripheral blood monocytes differentiating and maturing into dendritic cells. Methods Human peripheral blood monoeytes were obtained by discontinuous density gradient centrifuged method. Monoeytes of different groups （cytokine group, saikoside group, co-culture group）were cultured with different stimulating factors such as GM-CSF, IL-4, TNF-α and saikoside in vitro. The inverted phase contrast microscope, Wright＇ s staining, immunocytochemical staining were used respectively to observe the changes of morphology, surface differentiation antigen including CD14, CD83, HLA-DR, S-100, and the expression of intracellular transcription factor NF-κB p50 protein, in the process of monocytes differentiating into dendritic cells. The image analysis and SPSS software were used to analyze the results. Results The obtained monoeytes and dendritic cells possessed their respective cell shape and cell surface antigen marker by cell morphology observation and cell surface marker immunoeytoehemical staining such as CD14, HLA-DR, S-100 and CD83. The monoeytes differentiated into immateure dendritic cells（imDC） when cultured with RPMI-1640 medium containing GM-CSF and IL-4 for 7 days. The imDC differenfiatd into mature dendritic cells （mDC） when cultured with RPMI-1640 medium containing GM-CSF, IL-4 and TNF-a for another 3 days. The expresion of NF-κB p50 in the two cells showed a significant difference （P 〈 0.05） . When Monocytes were cultured with RPMI-1640 medium containing saikoside a only for 7 days, NF-κB p50 staining in their nucleus was brown-yellow, but differentiated imDCs was not found. As monocytes were cultured with RPMI-1640 medium containing GM-CSF, IL-4 and saikoside for 7 days, the cells were identified as imDCs by immunocytochemical staining. Then the maturation promoting factor TNF-α was added, cultured imDC for another 3 days, the nucleus appeared obvious NF-κB p50 brown-yellow staining which was stronge

关 键 词：树突状细胞 细胞表面抗原 核转录因子-κB P50 柴胡皂苷A 免疫细胞化学 人 

分 类 号：R392[医药卫生—免疫学]