不同方法对未成熟树突状细胞体外诱生的研究

Comparative research on protocol used in inducing immature dendritic cells from rat bone marrow progenitors

目的筛选最佳体外诱生大鼠未成熟树突状细胞(imDC)的方法。方法用低剂量(0.2ng/mL)粒细胞巨噬细胞集落刺激因子(recombination rat GM-CSF,rrGM-CSF)体外诱导imDC;用常规剂量(5ng/mL)的GM-CSF加大鼠白细胞介素-4(recombinationratIL-4,rrIL-4)(5ng/L)体外诱导imDC;rrGM-CSF+rrIL-4再加大鼠白细胞介素-10(recombination rat IL-10,rrIL-10)(10ng/mL)体外诱导imDC;第13天收获细胞,观察形态;流式细胞仪检测表面抗原;混合淋巴细胞培养(MLR)检测DC刺激同种异体T淋巴细胞的能力,培养上清白细胞介素-12(IL-12)的浓度和细胞增殖情况等方面进行考察,对比筛选。结果低剂量GM-CSF组和IL-10组DC与GM-CSF+IL-4组DC相比,符合imDC的特点;GM-CSF+IL-4组DC表现成熟DC的特点,但细胞增殖情况低剂量GM-CSF组明显低于GM-CSF+IL-4+IL-10组,两组相比差别具有显著性。结论GM-CSF+IL-4+IL-10组是最佳诱导imDC的方法。

[Objective] To select the optimal protocols from several protocols used in inducing immature dendritic ceils from Rat bone marrow （BM） progenitors. [Methods] The first protocol： immature dendritic cells（GMlo DC） were generated with low concentrations of granulocyte-macrophage colony-stimulating factor （GM-CSF） in the absence of the other cell factors in vitro ; the second protocol： immature dendritic cells were generated with conventional method with routine concentration （5 ng/mL） of GM-CSF plus intedeukin-4 （5 ng/L）; the third protocols ： immature dendritic cells were generated with routine coneentration （5 ng/mL） of GM-CSF, intedeukin-4 （5 ng/L） plus intedeukin-10; ImDC was induced simultaneously and then harvested on day 13. The phenotypic and functional properties of imDC induced with three protocols were compared with each other. Intedeukin-12 concentrations of supernatant and cell yield in three protocols .were investigated. [Results] Compared to DC induced with the second protocol, DC induced with the third protocol and the first protocol were phenotypically immature, weak stimulators of allogeneic and peptide-specific T cell responses. However, DC induced with the second protocol was phenotypically mature. Cell yield in the first protocol was significantly lower than the yield in the third protocol and the second protocol. [Conclusion] The protocol with GM-GSF plus IL-4 plusIL-10 could generate large number of imDC from rat bone marrow in vitro is optimal protocol.