“DC细胞工厂”——脐带血CD34+造血干细胞来源的DC的大量制备
| 作者: |
1,2刘刚,
1,2李康,
1,2董建涛,
1,2蔡建辉
1 河北医科大学，石家庄050017 2 河北省人民医院， 石家庄 050051 |
| 通讯: | |
| DOI: | 10.3978/j.issn.2095-6959.2015.06.S133 |
摘要
背景：树突状细胞(Dendritic Cell，DC)充当 着免疫系统中的“警察”角色，它能够识别体内异常 抗原信息并通过激活初始T细胞而诱导特异性的免疫 应答。以DC为基础制备的肿瘤疫苗能有效的构筑抗 肿瘤免疫屏障，因此DC被认为是充满前景的免疫治 疗工具。作为原料细胞，大量成熟，功能均一的DC细胞是制备DC疫苗的必须条件，而临床上广泛应用 的DC细胞来自于自体外周血，数量在106~108之间， 无法充分满足临床应用。因此，探索新的大量制备 DC的方法成为临床上迫切的需求。目的：本实验旨 在探索新的由CD34+造血干细胞大量诱导DC细胞生 成的方法，并通过体外实验证实这种CD34-DC肿瘤 疫苗相比外周血单核细胞来源的DC疫苗(PBMC-DC) 具有类似的或者更好的诱导抗肿瘤免疫应答的能力。 方法：分离脐带血CD34+造血干细胞并利用GM-CSF/ SCF进行连续30 d的扩增培养，期间通过贴壁持续收 获DC。外周血单核细胞诱导的DC作为对照。对比两 种来源的DC形态，表型和包括抗原吞噬，混合淋巴 增殖，细胞因子分泌，趋化能力的功能特征。分别将 其制备成肿瘤DC疫苗，并对比体外诱导的CTL IFN-γ 分泌能力和针对肿瘤靶细胞的细胞毒性。结果：1) GM-CSF/SCF细胞因子组合连续培养CD34+细胞30 d 后，共收获~1010总细胞量和~109的DC。与PBMCDC 相比，具有类似的形态特征；2)成熟状态时， CD34-DC与PBMC-DC具有类似的细胞表型(CD80， CD83，CD86，HLA-DR)，而未成熟时CD34-DC表 型好于PBMC-DC，尤其是CD80表达较早上调；3)无 论是新鲜的还是冻存后复苏的，CD34-DC和PMBCDC 在GM-CSF/IL-4培养的第5天吞噬功能最强，是抗 原负载的最佳时间窗；4)鸡尾酒式细胞因子作用下， CD34-DC较PBMC-DC具有更强的IL-12p70和IL-10的 分泌能力，且前者诱导的CTL IFN-γ分泌水平更高， 同时5)其表现出了更强的细胞毒作用；6)正常健康 孕妇脐带血中CD34+造血干细胞具有稳定的增殖和向 DC分化的潜能。结论：我们建立了一套由脐带血造 血干细胞诱导大量DC生成的方法并称之为“DC工 厂”。由其制备的DC抗肿瘤疫苗是将来能够用于临 床的充满前景的免疫刺激剂。
关键词：
树突状细胞
肿瘤疫苗
CD34+造血干细胞
制备
DC factory: a new source of DC derived from CD34+ progenitor cells
DOI: 10.3978/j.issn.2095-6959.2015.06.S133
Abstract
Background: Dendritic cell (DC) serves as “police” to recognize abnormal antigen and elicit potent adaptive immune responses by activating naïve T lymphocytes, and it is a promising immune tool in from of DC-based vaccine which is effective in building anti-tumor immune barrier. Large amount of matured, functional, homogeneous DC are essential for DC vaccination or DC-induced immunotherapy, but number of autologous DC from cancer patients ranged from 106~108 that is insufficient for clinical use. Thus, exploration of new method of DC generation is urgently needed. Objective: To explore a new method of DC generation from CD34+ progenitors in fresh cord blood and identify the novel CD34-DC vaccine has comparable or superior capacity in inducing immune responses ex vivo compared with conventional PBMC-DC vaccine. Methods: Isolation of CD34+ progenitors of cord blood and cultured them in GM-CSF/SCF medium for cell expansion for continuously 30 days, and DC were harvested by plastic adherent every three days during the whole expansion period. DC derived from peripheral blood monocytes were served as controls. Both of them were compared in morphology, phenotype, and functional assays including antigen uptake, mixed leukocyte reaction, cytokine secretion and chemotaxis. And in addition, DC vaccine was prepared with tumor lysate loading and the IFN-γ, cytotoxicity of CTL was compared. Results: 1) GM-CSF/SCF cytokines lead to ~ 10 log expansions of total CD34+ cells and ~ 9 logs of DCs during continuous 30 days cultivation ex vivo. CD34-DC exhibited similar features of morphology with PBMC-DC; 2) the expression rate of CD80, CD83, CD86 and HLA-DR in CD34-DC were comparable to PBMC-DC when matured, and in addition, CD80 expression in CD34-DC upregulated earlier; 3) CD34-DC exhibited stronger phagocytosis than PBMC-DC and the fifth culturing day in GM-CSF/IL-4 medium represented an ideal time points for antigen loading, even for those thawed from cryopreserved DCs in liquid nitrogen; 4) CD34-DC vaccine secreted higher levels of IL- 12 and IL-10 when stimulated by cocktailed cytokines than PBMC-DC, and the antigen-specific cytotoxic lymphocytes (CTLs) induced by them produced much more IFN-γ; 5) CD34-DC vaccine induced CTLs exhibited stronger cytotoxicity than those induced by PBMC-DC; 6) CD34+ progenitors in cord blood had stable amplification capability and differentiation potential to dendritic cells. Conclusion: Hence, a “DC factory” based on CD34+ progenitor enriched cord blood might be established, and its final production— DC vaccine, might be a promising immune stimulator for further use in cancer immunotherapy.