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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)悬液中单个核细胞(MNC)冷 冻保存后经不同诱导途径培养的树突状细胞(DC)的特性,探讨PBSC悬液冷冻保存后诱导培养DC的方法。方法:用 两种保护液-80℃冷冻保存PBSC悬液,分析复苏MNC中CD34+细胞、CD14+细胞、CD14+PI+细胞,并分两种方法诱 导培养复苏MNC:贴壁细胞经粒细胞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 前加入肿瘤坏死因子α(TNF-α)、布雷菲德菌素(BFA);非贴壁细胞经酪氨酸激酶3配体(FL)、干细胞因子(SCF)、GM -CSF、IL-4培养1周,再按前1种方法继续培养2周。培养结束后,流式细胞仪分析DC特性。结果:两组复苏 MNC与新鲜MNC比较:CD34+细胞含量均无明显差异(P>0．05);而CD14+细胞少,CD14+PI+细胞百分率高(P< 0．01);贴壁细胞少,贴壁细胞诱导培养DC效果不佳;非贴壁细胞扩增诱导均能获得大量成熟的激活的能够分泌IL -12(p40)的髓系DC。结论:PBSC悬液冷冻保存后能用于培养髓系DC,但培养方法应以扩增诱导为主。
AIM: To investigate the characteristics of dendritic cells (DCs) cultured in different pathways after cryopreservation of mononuclear cells (MNCs) from peripheral blood stem cell (PBSC) mobilized by granulocyte colony-stimulating factor (G-CSF) Methods of culturing DCs after cryopreservation of PBSC suspensions. Methods: The suspension of PBSC was stored at -80 ℃ in two protective solutions. The CD34 + cells, CD14 + cells and CD14 + PI + cells in MNC were resuscitated and cultured. The MNCs were cultured and resuscitated in two ways: adherent cells were transfected with granulocyte (GM-CSF) and interleukin-4 (IL-4) were cultured for 2 weeks. Tumor necrosis factor α (TNF-α) and brefeldin (BFA) were added before the end of culture. (FL), stem cell factor (SCF), GM-CSF and IL-4 for one week, then cultured for another two weeks according to the former method. After culturing, the DC characteristics were analyzed by flow cytometry. Results: There was no significant difference in the content of CD34 + cells between the two groups (P> 0.05). However, the number of CD14 + cells and the percentage of CD14 + PI + cells were higher (P <0.01) Adherent cells induced DCs to perform poorly; non-adherent cells could induce a large number of mature and activated myeloid DCs capable of secreting IL-12 (p40). Conclusion: PBSC suspension can be used to culture myeloid DC after cryopreservation, but the culture method should be mainly induced by amplification.