明胶法富集外周血单核细胞分化为树突状细胞的研究

发布时间：2018-06-06 16:36

本文选题：明胶 + 单核细胞　；参考：《山西医科大学》2012年硕士论文

【摘要】：目的：观察明胶法分离外周血单核细胞的效率以及将分离出的单核细胞刺激成熟为树突状细胞(dendritic cell, DC),观察其形态及表型特征,并与普通塑料粘附法对比。方法：使用人淋巴细胞分离液密度梯度离心法分离人外周血得到外周血单个核细胞(peripheral blood mononuclear cell, PBMC),根据培养瓶是否进行明胶包被分为明胶包被组(实验组)和普通塑料组(对照组)。均分外周血单个核细胞,按不同分组分离获得单核细胞。在重组人白细胞介素4(recombinant human interleukin-4, rhIL-4)和粒-巨噬细胞集落刺激因子(recombinant human granulocyte-macrophage colony-stimulating factor, rhGM-CSF)作用下刺激为非成熟树突状细胞,在促成熟鸡尾酒组合重组人白细胞介素1β (recombinant human interleukin-1β, rhIL-1β)、重组人白细胞介素6(recombinant human interleukin-6,rhIL-6)、重组人肿瘤坏死因子a (recombinant human tumor necrosis factor-a, rhTNF-a)和前列腺素E2(prostaglandin E2, PGE2)作用下诱导刺激为成熟树突状细胞。计数各组分离获得的单核细胞数。镜下观察对比两组血小板污染情况。流式细胞仪检测两组单核细胞的CD14、CD3、CD19阳性率,分别代表所得单核细胞的纯度、T、B淋巴细胞污染率。锥虫蓝拒染法计算单核细胞活率。单核细胞分化为树突状细胞后镜下观察对比两组所得树突状细胞的形态。使用流式细胞仪检测树突状细胞非成熟期和成熟期CDla,CD83的表达情况。结果：平均每组30ml外周血,普通塑料组和明胶包被组分别获得(12.3±3.56)和(15.8±3.05)×106个单核细胞,两组相比P0.05,差异有显著性意义,表明明胶法分离获得的单核细胞多于普通塑料粘附法。镜下观察到普通塑料组的血小板污染率明显高于明胶包被组。普通塑料组和明胶包被组所得单核细胞的CD14、CD3、CD19阳性率分别为(67.25±5.77)%和(81.56±5.83)%、(4.68±1.01)%和(2.89±0.81)%、(10.89±1.45)%和(7.68±1.54)%,各组相比P0.05,差异有显著性意义,表明明胶法获得的单核细胞的纯度大于普通塑料粘附法,T、B淋巴细胞污染率低于普通塑料粘附法。两组细胞活率分别为(94.6±1.58)%和(95.2±1.64)%,相比P0.05,差异无显著性意义,表明明胶法对细胞活力无明显不利影响。两组细胞刺激为树突状细胞后,均具有典型树突状细胞的形态学特征,非成熟期和成熟期表型CDla、CD83相比差异无显著性意义,认为明胶法对单核细胞刺激成熟为树突状细胞的功能无不利影响。结论：明胶法可以简单高效分离出外周血中单核细胞并成功刺激其分化为具有典型形态学及成熟表型的树突状细胞。
[Abstract]:Objective: to observe the efficiency of isolation of peripheral blood monocytes by gelatin method, and to observe the morphological and phenotypic characteristics of dendritic cells (DCN), which were stimulated by the isolated monocytes and matured into dendritic cells (DCN). Methods: human peripheral blood mononuclear cells (PBMC) were isolated from human peripheral blood by density gradient centrifugation method, and divided into gelatin according to whether the culture bottle was coated with gelatin. Coated group (experimental group) and ordinary plastic group (control group). Mononuclear cells were isolated from peripheral blood mononuclear cells in different groups. 4(recombinant human interleukin-4 (rhIL-4) and granulocyte-macrophage colony stimulating factor recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) stimulated immature dendritic cells. Recombinant human interleukin-1 尾, rhIL-1 尾, recombinant 6(recombinant human interleukin-6hIL-6, recombinant human tumor necrosis factor-a (rhTNF-a) and prostaglandin E2prostaglandin E2 (PGE2) were used to induce mature dendritic cells (dendritic cells) in the presence of a cocktail of prostaglandin 2 (PGE2), recombinant human interleukin-1 尾 (rhIL-1 尾), recombinant human interleukin (6(recombinant human) interleukin-6 (rhIL-6), recombinant human tumor necrosis factor-a (rhTNF-a) and prostaglandin E2 (PGE2). The number of monocytes isolated from each group was counted. The platelet contamination in the two groups was observed under microscope. The positive rate of CD14, CD3, CD19 in monocytes was detected by flow cytometry. The viability of monocytes was calculated by trypanosome blue exclusion method. The morphology of dendritic cells in the two groups was observed and compared with the differentiation of monocytes into dendritic cells. Flow cytometry was used to detect the CD83 expression of dendritic cells in immature and mature stages. Results: on average, 12.3 卤3.56) and 15.8 卤3.05) 脳 106 monocytes were obtained in each group of 30ml peripheral blood, ordinary plastic group and gelatin coated group, respectively. Compared with P0.05, the difference between the two groups was significant, indicating that the monocytes isolated by gelatin method were more than those by ordinary plastic adhesion method. The rate of platelet contamination in ordinary plastic group was significantly higher than that in gelatin coated group. The positive rates of CD14 and CD3 + CD19 were 67.25 卤5.77 and 81.56 卤5.83% and 2.89 卤0.81% and 10.89 卤1.45% and 7.68 卤1.54 of monocytes in the plastic group and gelatin coated group, respectively, and there was significant difference between the two groups (P0.05, P 0.05), and the positive rates of CD14 + CD3 + CD19 in the normal plastic group and gelatin coated group were 67.25 卤5.77% and 81.56 卤5.83%, respectively, and 2.89 卤0.81% and 7.68 卤1.54% in the normal plastic group and gelatin coated group, respectively. The results showed that the purity of monocytes obtained by gelatin method was higher than that by ordinary plastic adhesion method. The cell viability of the two groups was 94.6 卤1.58% and 95.2 卤1.64%, respectively. Compared with P0.05, there was no significant difference between the two groups, indicating that the gelatin method had no significant adverse effect on cell viability. After stimulated with dendritic cells, the two groups had the morphological characteristics of typical dendritic cells, and there was no significant difference in the phenotypes of CDla-CD83 between immature and mature stages. It is concluded that gelatin method has no adverse effect on the function of monocytes in stimulating maturation to dendritic cells. Conclusion: gelatin method can be used to isolate monocytes from peripheral blood and successfully stimulate their differentiation into typical morphology and formation. Mature phenotypic dendritic cells.
【学位授予单位】：山西医科大学
【学位级别】：硕士
【学位授予年份】：2012
【分类号】：R392

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