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目的 研究粒细胞集落刺激因子 (G CSF)动员的外周血干细胞 (PBSC)悬液中单核细胞和造血干/祖细胞分别诱导培养的树突状细胞 (DC)的特性 ,探讨临床应用PBSC悬液诱导DC的前景。方法 健康供者经G CSF动员后采集PBSC ,分两种方法诱导培养树突状细胞 :①贴壁细胞 (单核细胞 )经粒—单核细胞集落刺激因子 (GM CSF) +白细胞介素 4 (IL 4 )培养 2周 ,培养结束前 4 8、2 4h分别加入肿瘤坏死因子 (TNF α)、布雷菲德菌素 (brefeldinA ,BFA ) ;②非贴壁细胞 (含CD34+细胞 ) ,加入Flt3Ligand(FL) +干细胞因子 (SCF) +GM CSF +IL 4培养 1周 ,再按前一种方法继续培养 2周。培养结束后 ,流式细胞仪分析细胞表型和胞质 (c)IL 10 +(PE标记 )、cIL 12 (P4 0 ) +(TC标记 )细胞。结果 新鲜标本含CD14 +细胞 ( 18 4± 8 6 ) % ,CD34+细胞 ( 0 9± 0 4 ) %。以PBSC悬液中的 2× 10 6单个核细胞为起始细胞 ,前一种培养方法获得 ( 1 6± 0 6 )× 10 5细胞 ,细胞表型 :CD11c+( 97 3± 5 2 ) % ,CD86 +( 88 2± 6 8) % ,CD83+( 5 9 6± 8 4 ) % ,HLA DR+( 96 5± 7 1) % ,CD1a+( 36 6±7 5 ) % ,cIL 12 (P4 0 ) +( 15 9± 5 1) % ,cIL 10 +( 1 2± 0 4 ) % ;后一种培养方法获得 ( 1 2± 0 4 )× 10 6细胞 ,细胞表型
Objective To study the characteristics of dendritic cells (DCs) induced by monocytes and hematopoietic stem / progenitor cells from peripheral blood stem cell (PBSC) mobilized by granulocyte colony-stimulating factor (G CSF) and to investigate the clinical application of PBSC suspension Liquid induction of DC in the future. Methods The healthy donors collected PBSC after G CSF mobilization, and cultured dendritic cells in two ways: ① Adherent cells (monocytes) were stimulated by GM-CSF + IL-4 (IL 4) were cultured for 2 weeks. Tumor necrosis factor (TNFα) and brefeldin A (BFA) were added respectively 4, 8 and 24 hours before the end of culture. ② Nonadherent cells (including CD34 + cells) were added with Flt3Ligand (FL) + stem cell factor (SCF) + GM CSF + IL 4 for 1 week, followed by the previous method for 2 weeks. After culturing, the cell phenotype and cytoplasm (c) IL 10 + (PE marker), cIL 12 (P4 0) + (TC marker) cells were analyzed by flow cytometry. Results The fresh specimens contained 18 4 ± 8 6% of CD14 + cells and 0 9 ± 0 4% of CD34 + cells. The cells were cultured in the presence of 2 × 10 6 mononuclear cells in the PBSC suspension. The former culture method (16 ± 0 6) × 10 5 cells, the cell phenotype: CD11c + (97 3 ± 5 2)%, CD86 + (88 2 ± 6 8)%, CD83 + (596 ± 8 4)%, HLA DR + (96 5 ± 7 1)%, CD1a + (36 6 ± 7 5)%, cIL 12 (15 9 ± 5 1)% and cIL 10 + (1 2 ± 0 4)%, respectively. The latter culture method obtained (1 2 ± 0 4) × 10 6 cells,