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目的观察脐血来源的未成熟树突状细胞(imDC)低温冻存前后的生物学特性,探讨imDC的保存方法。方法取新鲜脐血分离单个核细胞(MNC),在体外经重组人粒细胞巨噬细胞集落刺激因子(rhGM-CSF)和重组人白细胞介素(rhIL)4诱导产生imDC后,加入二甲亚砜(DMSO)作为保护剂,-80℃降温,-196℃保存,40℃水浴复温,获得冻存imDC。光学显微镜下观察冻存的imDC与新鲜imDC形态,计算其锥虫蓝拒染回收率(TBR);流式细胞仪检测细胞表面成熟标志;混合淋巴细胞反应(MLR)检测细胞刺激未致敏T淋巴细胞的增殖能力。结果脐血MNC在rhGM-CSF和rhIL-4诱导下可向树突状细胞(DC)分化,相差显微镜显示细胞形态不规则,细胞表面呈树枝样突起;扫描电镜显示,细胞表面不规则,有树枝样突起和不规则皱褶。冻存imDC复苏后TBR为(86．8±1．3)％,冻存imDC在形态上与新鲜imDC无明显差异。MNC体外经rhGM-CSF和rhIL-4诱导生成的imDC表面CD1a阳性率为(62±8)％、CD14为(18±7)％、人类白细胞DR抗原(HLA-DR)为(67±5)％、CD80为(13±7)％、CD86为(12±5)％;反映DC成熟的表面标志物CD83为(4．6±2．0)％,符合imDC的表型特征。冻存imDC的CD80、CD86、CD83分别为(15±5)％、(17±5)％、(7．4±3．3)％,较新鲜imDC有所增高(P<0．05),但仍符合imDC的表型特征。对照组MLR的每分钟放射性荧光闪烁计数(cpm)值为(488±197)min-1;新鲜imDC组为(463±104)min-1,与冻存imDC组的cpm值(512±78)min-1比较,差异无统计学意义(P>0．05)。各组细胞刺激指数(SI)均<2。新鲜imDC和冻存imDC均不能有效刺激同种未致敏T淋巴细胞增殖。结论本实验中获得的冻存imDC具有足够的细胞活力,其细胞免疫表型和细胞功能具有不成熟特征,说明利用DMSO低温保存imDC的方法可行。
Objective To observe the biological characteristics of imDC dendritic cells (imDC) cryopreserved before and after cryopreservation, and to investigate the preservation methods of imDC. Methods Mononuclear cells (MNCs) were isolated from fresh umbilical cord blood. After imDC was induced by recombinant human granulocyte-macrophage colony-stimulating factor (rhGM-CSF) and recombinant human interleukin (rhIL) 4, Sulfone (DMSO) as a protective agent, cooled at -80 ° C, stored at -196 ° C and rewarmed at 40 ° C in a water bath to obtain imDC. The morphological changes of imDC and fresh imDC were observed under an optical microscope. The TBR of trypan blue exclusion was calculated. Flow cytometry was used to detect the cell surface maturation markers. Mixed lymphocyte reaction (MLR) Lymphocyte proliferation ability. Results Umbilical cord blood MNCs could differentiate into dendritic cells (DCs) under the induction of rhGM-CSF and rhIL-4. The morphological changes of the dendritic cells (DCs) were observed by phase contrast microscopy. The cell surface was dendritic. Scanning electron microscopy showed that the cell surface was irregular. Branches and irregular folds. The TBR after imDC resuscitation was (86.8 ± 1.3)%, while imDC in cryopreservation was not significantly different in morphology from fresh imDC. The positive rate of CD1a on imDC surface induced by rhGM-CSF and rhIL-4 was (62 ± 8)% in MNC, (18 ± 7)% in CD14 and (67 ± 5) %, CD80 was (13 ± 7)% and CD86 was (12 ± 5)%; CD83, a marker of DC maturation, was (4.6 ± 2.0)%, consistent with the imDC phenotypic characteristics. The percentage of CD80, CD86 and CD83 in imDC were (15 ± 5)%, (17 ± 5)% and (7.4 ± 3.3)%, respectively, But still in line with imDC phenotypic characteristics. (488 ± 197) min-1 per minute in control group, (463 ± 104) min-1 in fresh imDC group, and 512 ± 78 in cryogenic imDC group min-1, the difference was not statistically significant (P> 0.05). The cell stimulation index (SI) of each group was less than 2. Neither imDC nor imDC can effectively stimulate the proliferation of the same kind of non-sensitized T lymphocytes. Conclusion The imDC obtained in this experiment has sufficient cell viability, immature characteristics of its cellular phenotype and cell function, indicating that it is feasible to use imd DMSO to preserve imDC.