粒细胞集落刺激因子动员的外周血干细胞悬液诱导培养树突状细胞的研究

A study on dendritic cells cultured from granulocyte colony-stimulating factor-mobilized peripheral blood stem cell harvests

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 。

Objective To assess the characters of the dendritic cells (DCs) induced by suitable methods from granulocyte colony stimulating factor(G CSF) mobilized peripheral blood stem cell (PBSC) harvests.Methods The non adherent cells were cultured for 1 week in FL+SCF+GM CSF+IL 4,and the adherent cells in PBSC harvests were cultured for 2 weeks in the presence of GM CSF+IL 4.DC markers were analyzed using flow cytometry.Intracellular cytokine staining and the staining of IL 12 (P40) and IL 10 were used for determining the proportion of IL 12 producing DC and IL 10 producing DC in the cultured DCs.Results PBSC harvests contained CD14 + cells (18.4±8.6)%,CD34 + cells (0 9±0 4)%.The DCs cultured from the adherent cells and the non adherent cells expressed CD11c + (97 3±5 2)% vs (91 4±6 4 )%,CD86 + (88 2±6 8)% vs (76 8±6 9)%,CD83 + (59 6±8 4)% vs (47 3±9 3)%,HLA DR (96 5±7 1)% vs (65.3±11.3)%,CD1a + (36.6±7.5)% vs (48 2±5 9)%,cIL 12 (P40) + (15 9±5 1)% vs (14 8±4 2)%,cIL 10 (1 2±0 4)% vs (1 4±0 5)%.Conclusion G CSF mobilized PBSC harvests contain more monocytes and hematopoietic stem cells/progenitors,and they can be induced to cultivate mature,activated DC which can secrete IL 12 (P40);making hematopoietic stem cells/progenitors receive a culture process of amplification first and inducing differentiation afterwards may gain more DC;in case of needing large amount of autologous DC for treatment,G CSF mobilized PBSC suspension is a very good material for inducing and cultivating DC.