脐血来源未成熟树突状细胞低温冻存的实验研究

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。

Experimental study on cryopreservation of immature dendritic cells derived from cord blood

OBJECTIVE: To investigate the biological properties of immature dendritic cells( imDC) derived from cord blood before and after cryopreservation, so as to provide a method for preservation of imDC. METHODS: Immature dendritic cells were generated from human cord blood (CB) monocytes and cultured with rhGM-CSF and rhIL-4, and 10% DMSO was added into culture medium as cryopreservation reagent. After freezing in - 80 degrees C refrigerator, the cells were finally cryopreserved in - 196 degrees C liquid nitrogen, and then thawed with 40 'C water, and they were finally named frozen-thawed imDC. The morphology of imDC were observed with light microscope, and TBR were calculated. Cellular surface markers for DC maturation were determined with flow cytometry, and the ability of the cells to stimulate proliferation of non-sensitized T lymphocyte was determined with allogenetic mixed lymphocyte reaction. RESULTS: Monocyte (MNC) from cord blood could differentiate into DC after GM-CSF and rhIL-4 induction. Under light microscope, the cells showed irregular morphology, with branch-like prominence on the cell surface. Similar changes were also observed with scan electron microscope. The cryopreserved imDC were resistant to trypan blue staining, and TBR was (86. 8 +/- 1. 3) % . There was no obvious difference in the cell morphology between cryopreserved and fresh imDCs. The expression of cell surface markers and maturation markers in imDCs before cryopreservation were as follows: CDla(62 +/-8)% , CD14 (18 +/- 7)% , HLA-DR (67 +/- 5)% , CD80 (13+/-7)%, CD 86 (12+/- 5) % . Though the expression of CD80, CD86 and CD83 of cryopreserved imDC increased to (15 +/-5)% , (17 +/-5)% and (7.4 +/-3. 3)% , respectively( P <0.05), they still possessed the phenotype of imDC. There exhibited no obvious difference in cmp value between fresh imDC[ (463 +/- 104) min(-1) ] and cryopreserved imDC[ (512 +/-78 )min(-1) ] , ( P > 0. 05 ). The cpm in control group was (488 +/- 197 ) min'. The stimulation index in all groups was lower than 2, and both fresh imDC and cryopreserved imDC could not stimulate the proliferation of non-sensitized T lymphocyte. CONCLUSION: The cryopreserved imDC exhibits immature characteristic in cell phenotypes, function and good cell activity, indicating that the method of cryopreservation of imDC is feasible.