人脐血树突状细胞体外培养及免疫功能的研究

发布时间：2018-09-10 12:07

【摘要】： 目的通过人脐血树突状细胞(Dendritic cells,DC)的体外培养,对DC进行形态学观察及免疫表型检测,探讨其对抗肿瘤效应细胞CTL活性的影响。方法本研究首先在无菌条件下常规采集脐血,用淋巴细胞分离液分离脐血单个核细胞(cord blood mononuclear cell,CMNC)。将获得的单个核细胞(mononuclear cell,MNC)分为7组,每组加入3ml的RPMI-1640培养基,分别添加不同的细胞因子组合诱导DC的生成。其中一组为对照组,不加任何细胞因子培养;另外6组分别添加rhIL-4和rhGM-CSF,其中3组在第五天再分别添加TNF-α。14d后,收集贴壁DC细胞,分别用流式细胞技术检测CD83、CD86、CD14、HLA-DR等免疫表型。用MTT比色法检测DC活化的T细胞与对照组细胞(异基因的淋巴细胞组)对人红白血病细胞系K562细胞的杀伤作用。结果 7组CMNC培养14天后,流式细胞技术检测贴壁细胞中第六实验组(1000u/ml的rhIL-4和1000u/ml的rhGM-CSF,培养5天后加入TNF-α50u/ml)的HLA-DR,CD14, CD83和CD86高表达,与其他组相比较P0.05,CMNC诱导培养生成DC细胞的含量为,6组5组4组3组2组1组7组。应用MTT比色法测定从脐血诱导分化的DC活化的CTL细胞能特异杀伤K562肿瘤细胞,杀伤作用远大于对照组(p0.05),在效靶比为10:1时杀伤作用最大(p0.01),MTT比色法测得值为28.97±6.62%,提示杀伤率随效靶比的增加而增加。结论 CMNC可在体外经细胞因子组合诱导成为DC,其中在1000u/ml的rhIL-4和1000u/ml的rhGM-CSF,培养5天后加入TNF-α50u/ml的条件下诱导DC的数量最多;rhIL-4、rhGM-CSF和TNF-α不仅可以促进细胞形态的生成,而且明显增加DC生成数量。并且DC在体外能诱导产生抗人红白血病细胞系K562细胞毒效应。
[Abstract]:Objective to observe the morphology and immunophenotype of human umbilical cord blood dendritic cells (Dendritic cells,DC) in vitro and to investigate the effect of DC on the CTL activity of tumor effector cells. Methods Cord blood was collected under aseptic condition and (cord blood mononuclear cell,CMNC was separated from cord blood mononuclear cells (cord blood mononuclear cell,CMNC). The mononuclear cells (mononuclear cell,MNC) were divided into 7 groups. Each group was added with RPMI-1640 medium of 3ml, and different cytokine combinations were added to induce the formation of DC. One group was the control group, without any cytokine culture, the other six groups were added rhIL-4 and rhGM-CSF, respectively, and 3 groups were added TNF- 伪路14 d after the fifth day, the adherent DC cells were collected, and the immunophenotypes of CD83,CD86,CD14,HLA-DR were detected by flow cytometry. The cytotoxicity of T cells activated by DC and control cells (allogeneic lymphocyte group) on human erythroleukemia cell line K562 was detected by MTT colorimetry. Results after 14 days of CMNC culture in 7 groups, the high expression of HLA-DR,CD14, CD83 and CD86 in the sixth experimental group (rhIL-4 of 1000u/ml and rhGM-CSF, of 1000u/ml) were detected by flow cytometry after adding TNF- 伪 50u/ml for 5 days. Compared with other groups, the content of P0.05-CMNC-induced DC cells was as follows: 6 groups, 5 groups, 4 groups, 3 groups, 2 groups, 1 group, 7 groups. K562 tumor cells were specifically killed by DC activated CTL cells induced from cord blood by MTT colorimetric assay. The killing effect was much greater than that in the control group (p0.05), and the maximum killing effect (p0.01) was obtained by MTT colorimetry at 10:1, indicating that the killing rate increased with the increase of the effect-target ratio. Conclusion CMNC can be induced into DC, by cytokine combination in vitro. The number of DC induced by TNF- 伪 50u/ml is the most in 1000u/ml rhIL-4 and 1000u/ml rhGM-CSF, culture for 5 days. RhIL-4 rhGM-CSF and TNF- 伪 can not only promote the formation of cell morphology. Moreover, the amount of DC production was significantly increased. Moreover, DC can induce cytotoxic effect of human erythroleukemia cell line K562 in vitro.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2006
【分类号】：R392

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