树突状细胞体外诱导方案的对比

发布时间：2018-04-21 14:09

本文选题：树突状细胞 + 体外诱导　；参考：《细胞与分子免疫学杂志》2015年12期

【摘要】：目的探讨并比较不同细胞因子组合、诱导时间对树突状细胞(DC)体外诱导、成熟,分泌细胞因子水平和刺激淋巴细胞增殖能力的影响,为建立DC体外高效、规模化培育体系提供依据。方法分离健康人外周血单个核细胞(PBMC),贴壁法获得DC前体细胞,用白细胞介素4(IL-4)和粒细胞巨噬细胞集落刺激因子(GM-CSF)诱导2 d,将其分为6组。A组加入肿瘤坏死因子α(TNF-α)、IL-1α和前列腺素E2(PGE2);B组加入α干扰素(IFN-α);C组加入脂多糖(LPS);3组细胞继续培育2 d。D~F组细胞先进行1/3体积换液,24 h后,分别再加入上述A~C组的成熟因子,再继续诱导2 d。分别收获各组上清和细胞,进行细胞计数,采用流式细胞术进行表型分析;采用ELISA检测各组DC分泌IL-12p70的水平、采用细胞增殖检测试剂盒刺激淋巴细胞增殖的能力。结果 6组体外诱导体系均能培育出形态学类似DC的细胞,DC数量与纯度(均达到96%以上),各组间无显著性差异。A组与D组比较:CD40、CD80、CD83、CD86共刺激分子表达水平相近,但D组DC分泌IL-12p70水平高于A组;B组与E组比较:E组DC的CD86表达水平显著高于B组,CD80表达量略高于B组,2组DC分泌IL-12p70的水平相近;C组与F组比较:CD40、CD80、CD83、CD86共刺激分子表达水平和分泌IL-12p70的水平均相近。6组DC刺激淋巴细胞增殖水平均相近。三种不同成熟因子(组合)比较:B组DC共刺激分子CD80水平明显低于C组和A组,且IL-12p70分泌水平最低,而C组和A组共刺激分子表达水平与IL-12p70分泌水平相近。结论 TNF-α、IL-1α和PGE-2三种细胞因子联合以及LPS可有效地诱导DC表达高水平免疫共刺激分子、分泌高水平IL-12p70。
[Abstract]:Objective to explore and compare the effects of different cytokine combinations, induction time on the induction, maturation, secretion of cytokine levels and stimulation of lymphocyte proliferation in vitro of dendritic cells (DC), in order to provide a basis for the establishment of an efficient and large-scale culture system for DC in vitro, and to separate the peripheral blood mononuclear cells (PBMC) of healthy people before DC. Somatic cells were induced by interleukin 4 (IL-4) and granulocyte macrophage colony stimulating factor (GM-CSF) to induce 2 D, which were divided into 6 groups of.A groups with tumor necrosis factor alpha (TNF- alpha), IL-1 alpha and prostaglandin E2 (PGE2), B group adding interferon alpha (IFN- a), C group adding lipoprotein (LPS), and the 3 groups continued to cultivate 2 groups of cells to replace the volume of liquid first, After 24 h, the mature factors of the A~C group were added, and then 2 d. were further induced to harvest the supernatant and cells, the cell count, the flow cytometry was used for phenotypic analysis, the level of DC secretion of IL-12p70 was detected by ELISA, and the ability to stimulate the lymphocyte proliferation by the cell proliferation detection kit was used. The results of the 6 groups were induced in vitro. The number and purity of DC and purity (all up to 96%) could be produced in all conductors, and there was no significant difference between groups.A and D group: CD40, CD80, CD83, CD86 co stimulator level was similar, but D group DC secreted IL-12p70 level higher than that of A group. The level of DC secreted IL-12p70 in 2 groups was similar, and in group C and F group, CD40, CD80, CD83, CD86 co stimulatory molecules expression level and IL-12p70 level were similar to those of.6 group. The comparison of three different mature factors (combination) was significantly lower than that of the group and group. The secretory level of 12p70 is the lowest, while the expression level of CO stimulatory molecules in group C and A is similar to that of IL-12p70 secreting. Conclusion TNF- alpha, IL-1 alpha and PGE-2 three cytokine combinations and LPS can effectively induce high level immune costimulatory molecules in DC expression, and secrete high level IL-12p70..

【作者单位】：南京市胸科医院胸外科;南京市免疫细胞生物工程技术研究中心;南京市胸科医院呼吸内科;
【分类号】：R329.2

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