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目的:探讨经黄芪多糖诱生脐血来源的树突状细胞(DCs)体外介导的抗白血病细胞的细胞毒效应。方法:无菌条件下采集脐血,用淋巴细胞分离液分离获得脐血单个核细胞。分为两组,实验组:在含有浓度100mg/L黄芪多糖的10%胎牛血清的RPMI-1640完全培养液中培养;对照组:RPMI-1640完全培养液中培养。①在培养过程中用倒置光学显微镜和透射电镜观察细胞形态学变化;FCM技术检测培养第12天两组细胞的细胞免疫表型。②MTT比色法检测DCs对同种异体T细胞的增殖作用。③MTT比色法检测由DCs介导的对K562细胞的抑制作用。结果:①在培养的第72h后实验组细胞形态开始变化,随着培养时间的延长,树突状结构更加明显,第12天细胞呈典型的树突状细胞形态;对照组细胞生长缓慢,培养至第12天细胞呈梭形巨噬细胞形态。培养至10d的实验组细胞透射电镜下呈典型的树突状细胞形态。培养12天实验组细胞高表达DCs特异性抗原CD1a、CD80、CD83和CD86,与对照组对应比较差异均有显著性(P<0.01)。②在混合淋巴细胞反应中,经黄芪多糖诱生的DCs具有明显激发同种异体T淋巴细胞增殖的能力,且随数量的增加而作用增强。③经黄芪多糖诱生的DCs负载肿瘤抗原后可诱导肿瘤特异性CTL产生抗白血病细胞毒效应,而对照单独T细胞对K562细胞的杀伤作用很弱,各对应效靶比实验组和对照组之间相比有显著的统计学意义(P<0.05或0.01);各实验组间比较差异显著(P<0.05或0.01);各对照组之间比较无统计学意义(P>0.05)。结论:①黄芪多糖可诱导脐血来源的单核细胞(即DCs前体细胞)定向分化为功能性(即成熟)DCs;②黄芪多糖诱生的DCs具有强烈激发同种异体T淋巴细胞增殖的能力,且随数量的增加而作用增强;③黄芪多糖诱生脐血来源的DCs可负载肿瘤抗原诱导特异性CTL的产生,发挥显著的抗白血病细胞毒效应。
Objective: To investigate the cytotoxic effect of dendritic cells (DCs) induced by astragalus polysaccharides on cord blood-derived anti-leukemia cells in vitro. Methods: Umbilical cord blood was collected under aseptic conditions, and cord blood mononuclear cells were isolated by lymphocyte separation. Divided into two groups, the experimental group: RPMI-1640 complete medium containing 10% fetal bovine serum with 100mg / L APS; control group: RPMI-1640 complete culture medium. ①In the course of culturing, morphological changes of cells were observed by inverted optical microscope and transmission electron microscope; Cell immunophenotypes of the two groups were detected by FCM. ② MTT colorimetric assay DCs proliferation of allogeneic T cells. ③ MTT colorimetric assay by DCs-mediated inhibition of K562 cells. Results: (1) The morphology of the experimental group began to change after 72 h of culture, and the dendritic structure was more obvious with the extension of culture time. On the 12th day, the morphology of the dendritic cells was typical. The cells in the control group grew slowly and cultured By day 12, the cells were spindle-shaped macrophages. The cultured cells cultured to 10 days showed typical dendritic cell morphology under transmission electron microscope. The cultured DCs specific antigens CD1a, CD80, CD83 and CD86 of the 12-day experimental group were significantly different from those of the control group (P <0.01). ② In the mixed lymphocyte reaction, DCs induced by Astragalus polysaccharide could obviously stimulate the proliferation of allogeneic T lymphocytes, and increased with the increase of the number. ③ DCs induced by astragalus polysaccharide could induce tumor-specific CTL to produce anti-leukemia cytotoxic effect, whereas control T cells alone had a weak cytotoxic effect on K562 cells. The response targets of each group were significantly lower than those of experimental group and control group (P <0.05 or 0.01). There was a significant difference between the experimental groups (P <0.05 or 0.01). There was no significant difference between the control groups (P> 0.05). Conclusion: Astragalus polysaccharides can induce the differentiation of umbilical cord blood mononuclear cells (DCs precursor cells) into functional (ie, mature) DCs; (2) DCs induced by Astragalus polysaccharide can strongly stimulate allogeneic T lymphocyte proliferation Ability and increased with the increase of the quantity; (3) Cordyceps polysaccharide induced cord blood-derived DCs can load tumor antigen-induced specific CTL production, play a significant anti-leukemia cytotoxic effect.