改良细胞因子鸡尾酒法对树突状细胞诱导的研究

发布时间：2018-04-11 02:15

  本文选题：树突状细胞 + 细胞因子鸡尾酒　； 参考：《安徽医科大学》2013年硕士论文

【摘要】：目的：探讨优化人γhG-CSF动员的外周血采集物来源的树突状细胞（dendritic cell，DC）成熟诱导条件，以便制备更有效的DC疫苗应用于临床，观察并比较不同诱导条件下制备的DC疫苗在体外特异性抗肿瘤免疫反应的能力。 方法：利用密度梯度离心法从重组人粒细胞集落刺激因子（γhG-CSF）动员的外周血采集物分离出单个核细胞，以重组人粒细胞-巨噬细胞集落刺激因子(γhGM-CSF)和重组人白细胞介素-4(γhIL-4)体外诱导成不成熟树突状细胞（imDC），分别采用传统细胞因子鸡尾酒（IL-1β、IL-6、TNF-α、PGE2）和改良细胞因子鸡尾酒（IL-1β、IL-6、TNF-α、polyI:C、CpG ODN）刺激成熟，并设有对照组（只加IL-4、GM-CSF）。3d后收获成熟DC，观察细胞形态、流式细胞仪检测DC的内吞能力通过异硫氰酸荧光素标记的葡聚糖（FITC-Dextran）、检测DC表面CD83、CD1a、CD80、HLA-DR、CD86和CCR-7，酶联免疫吸附法（ELISA）检测其IL-12、IL-10的分泌，MTT法检测其刺激淋巴细胞增殖能力。实验中三组DC分别转染A549细胞的总RNA，酶联免疫吸附法（ELISA）检测IFN-γ的分泌，乳酸脱氢酶(LDH)释放法测定杀伤肿瘤活性。 结果：传统细胞因子鸡尾酒（IL-1β、IL-6、TNF-α、PGE2）和改良细胞因子鸡尾酒（IL-1β、IL-6、TNF-α、polyI:C、CpG ODN）两种方法均能诱导DC成熟，以改良细胞因子鸡尾酒效果更佳，对照组几乎不诱导DC成熟。改良鸡尾酒方法诱导的成熟DC的FITC-Dextran内吞能力明显下降；DC成熟的表面标志物CD83、CD1a的表达率分别是84.44%、87.94%（P0.05）；IL-12分泌量明显增高（P 0.05）；IL-10分泌量明显增高（P 0.05）；成熟DC能有效的刺激T淋巴细胞增殖；转染A549细胞的总RNA后的改良组DC的IFN-γ分泌水平明显高于前两组（P 0.05），并能诱导肿瘤特异性CTL的产生进而杀伤肿瘤细胞。 结论：从经γhG-CSF动员的外周血采集物中利用密度梯度离心法分离诱导出具备典型形态和免疫表型的DC后，两种方法诱导DC成熟，实验证明改良细胞因子鸡尾酒法是诱导DC成熟的最佳方法。
[Abstract]:Objective: to study the maturation induction conditions of dendritic cells derived from peripheral blood samples mobilized by human 纬 hG-CSF in order to prepare more effective DC vaccine for clinical application.To observe and compare the specific anti-tumor immune response of DC vaccine prepared under different induction conditions in vitro.Methods: mononuclear cells were isolated from peripheral blood samples mobilized by recombinant human granulocyte colony stimulating factor (纬 hG-CSF) by density gradient centrifugation.Immature dendritic cells (imDCA) were induced by recombinant human granulocyte-macrophage colony stimulating factor (纬 hGM-CSF) and recombinant human interleukin-4 (纬 hIL-4) in vitro. The mature cells were stimulated by traditional cytokine IL-1 尾 -IL-6TNF- 伪 PGE2 and modified cytokine cocktail IL-1 尾 IL-6TNF- 伪 polyCCpG ODN.The control group (IL-4 + GM-CSFN. 3 d) was used to harvest the mature DCs and observe the morphology of the cells.The endocytosis of DC was detected by flow cytometry by FITC-Dextranan labeled with fluorescein isothiocyanate, HLA-DRCD86 and CCR-7 by CD83T CD1a1 + CD80 on the surface of DC, and IL-12 IL-10 by MTT assay by enzyme linked immunosorbent assay (Elisa).In the experiment, the total RNAs of A549 cells transfected with three groups of DC were detected by enzyme linked immunosorbent assay (Elisa) to detect the secretion of IFN- 纬, and the activity of killing tumor was determined by lactate dehydrogenase (LDH) release method.The FITC-Dextran endocytosis of mature DC induced by modified cocktail method was significantly decreased. The expression rates of CD83- CD1a, the surface marker of DC maturation, were 84.444.444.44 / 87.94A and 87.94A respectively. The secretion of IL-12 was significantly increased in mature DCs, and the secretion of IL-10 was significantly increased in mature DCs, and the proliferation of T lymphocytes was effectively stimulated by mature DCs.The level of IFN- 纬 secretion in the modified DC after total RNA transfection was significantly higher than that in the former two groups (P 0.05), and it could induce the production of tumor-specific CTL and then kill the tumor cells.Conclusion: dendritic cells with typical morphology and immunophenotype were isolated from peripheral blood samples mobilized by 纬 hG-CSF by density gradient centrifugation.It is proved that the modified cytokine cocktail method is the best method to induce DC maturation.
【学位授予单位】：安徽医科大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R392

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