细胞因子对外周血树突状细胞的体外诱导成熟及其功能影响

发布时间：2018-07-23 17:09

【摘要】：[目的] 选用几种不同的细胞因子组合,体外培养人外周血单个核细胞,诱导树突状细胞(dendritic cells,DCs)产生,并检测其表面分子的表达及刺激同种异体T淋巴细胞增殖的功能,从而建立诱导树突状细胞的最佳实验方法。并探讨细胞因子对DCs体外诱导、成熟及其功能的影响。 [方法] 无菌采集健康人外周血,肝素钠抗凝,采用密度梯度离心的方法获得单个核细胞,用含10%胎牛血清的RPMI1640培养基在24孔板上贴壁培养2小时,弃去未贴壁细胞,即获得贴壁细胞,在24孔板上用含以下6组不同细胞因子的RPMI1640培养基进行培养①对照组;不加细胞因子②rhGMCSF/rhIL-4③rhGM-CSF/rhIL-4/PHA④rhGM-CSF/rhIL-13⑤rhGM-CSF/rhIL-13/PHA⑥PHA,各利因子加入的剂量分别为(在培养液中的最终浓度):rhGM-CSF(50ng/ml)、rhIL-4(40ng/ml)、rhIL-13(40ng/ml)、PHA(50μg/ml),观察DC的生成及免疫表型的表达情况。6天后,分别在各中加入LPS、IL-2、TNF-α等促成熟因子诱导DC成熟,加入的剂量分别为:LPS(20ng/ml)、IL-2(5ng/ml)、TNF-α(20ng/ml)镜下观察细胞形态。流式细胞术检测培养3,6,9天细胞CD1a、CD83、CD86、CD209表达:MTT法检测诱导培养的DC体外刺激同种异体T淋巴细胞增殖的活性,比较各成熟因子对DC作用的影响。 [结果] 1、经不同的细胞因子诱导培养6天后,倒置显微镜下可见多数细胞呈悬浮生长,细胞胞体较大,细胞表面可见有不规则突起。2、存IL-4、GM-CSF组合中加入刺激剂TNF-α、LPS或IL-2后,DC表面CD1a、CD83、CD86、CD209表达明显升高,其刺激同种异体T淋巴细胞的能力明显增强,刺激细胞/反应细胞为1:10时刺激T细胞增殖能力最明显。在各组中,尤以TNF-α、
[Abstract]:[objective] Human peripheral blood mononuclear cells (PBMC) were cultured in vitro with several different cytokines to induce the production of dendritic cells (DCs), and to detect the expression of surface molecules and the function of stimulating the proliferation of allogeneic T lymphocytes. Thus the best experimental method for inducing dendritic cells was established. The effects of cytokines on the induction, maturation and function of DCs in vitro were investigated. [methods] Mononuclear cells were obtained by density gradient centrifugation. Mononuclear cells were collected from healthy human peripheral blood and treated with heparin sodium. The mononuclear cells were cultured on 24-well plate for 2 hours on RPMI1640 medium containing 10% fetal bovine serum, and the unadherent cells were discarded. The adherent cells were obtained and cultured on the 24-well plate in RPMI1640 medium containing the following 6 groups of cytokines. Without adding 2rhGMCSF / rhIL-43rhGM-CSF / rhIL-4 / PHA4rhGM-CSF / rhIL-135rhGM-CSF / rhGM-CSF / PHA6PHAs, the dosage of each factor was (the final concentration in the culture medium): (the final concentration of rhGM-CSF (50ng/ml), rhIL-4 (40ng/ml), rhIL-13 (40ng/ml), PHA (50 渭 g/ml), respectively), the generation of DC and the expression of immunophenotype were observed 6 days after the addition of rhGM-CSF / rhIL-43rhGM-CSF / rhIL-4rhGM-CSF / rhIL-13rhGM-CSF / rhGM-CSF / rhIL-13rhGM-CSF / PHA6PHA. The maturation of DC was induced by adding LPS-IL-2TNF- 伪, the dosage of which was 20ng/ml (20ng/ml) / IL-2 (5ng/ml) / TNF- 伪 (20ng/ml) respectively. Flow cytometry was used to detect the expression of CD83A- CD83- CD86- CD209 and the effect of mature factors on the proliferation of allogeneic T lymphocytes induced by DC in vitro. [results] 1. After 6 days of induction and culture with different cytokines, most of the cells were found to be suspended and the cell bodies were larger under inverted microscope. There were irregular protrusions on the surface of the cells. The expression of CD1a- CD83, CD86, CD209, and the ability of stimulating allogeneic T lymphocytes were obviously increased after the addition of TNF- 伪 LPS or IL-2 to the combination of IL-4 and GM-CSF, and the expression of CD863 and CD86 + CD209 on the surface of the DC was significantly increased after the addition of the stimulator TNF- 伪 or IL-2. The T cell proliferation was the most obvious at 1:10. In each group, especially TNF- 伪,
【学位授予单位】：山东大学
【学位级别】：硕士
【学位授予年份】：2005
【分类号】：R392

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