人外周血树突状细胞体外稳定培养方法的建立及与磁珠法的比较

Establishment of in vitro stable culture of human peripheral blood dendritic cells and its comparation with magnetic activated cell sorting

目的 建立一种稳定的人外周血树突状细胞（DCs）体外培养的方法,并与磁珠分选法进行比较.方法 通过密度梯度离心法分离出志愿者的外周血单个核细胞（PBMC）,再分别应用磁珠分选法、贴壁法对PBMC进行培养,应用重组人集落刺激因子（rhGM-CSF）、重组人白细胞介素-4（rhIL-4）诱导获得DCs.倒置显微镜观察细胞形态变化,并分别在第3、5、6天用台盼蓝染色法进行细胞活力检测;经过1、2、5 h的贴壁培养后,应用流式细胞仪检测单核细胞表面CD14、CD1a、HLA-DR的表达以确定最佳贴壁时间;经人重组细胞因子诱导培养后,对所获得的细胞检测CD14、CD1a、CD86、CD83、HLA-DR的表达.采用同种混合淋巴细胞反应,评价DCs刺激T淋巴细胞增殖的能力.结果 经贴壁2 h后诱导培养的DCs形态较典型.磁珠分选法获得的DCs第5、6天细胞活力[（53.333±5.774）%、（38.333±7.638）%]明显低于第3天[（68.667±3.215）%,P均＜0.05];贴壁培养法获得的DCs第3、5、6天的细胞活力[（92.667±3.055）%、（94.000±1.000）%和（94.667±1.528）%]比较,差异无统计学意义（F=0.737,P＞0.05）;贴壁培养法获得的DCs第3、5、6天细胞活力均高于磁珠分选法（t值分别为9.374、12.021、12.527,P均＜0.05）.PBMC经磁珠分选前后CD14的阳性表达率分别为（32.457±12.351）%、（41.914±14.858）%,二者比较差异无统计学意义（t=1.295,P＞0.05）.单核细胞表面CD14的阳性表达率在培养2 h时[（35.267±4.658）%]高于培养1、5 h时[（15.033±6.189）%、（21.233±4.895）%,P均＜0.05].培养第6天,DCs表面CD14的阳性表达率[（2.200±1.356）%]较第1天[（32.328±14.517）%]明显下降（t=5.467,P＜0.05）,CD1a的阳性表达率[（43.371±16.250）%]较第1天[（12.300±6.223）%]显著升高（t=2.545,P＜0.05）;而CD86、CD83、HLA-DR的阳性表达率[（16.857±5.686）%、（9.343±5.230）%、（72.800±17.881）%]与第1天[（12.550±16.758）%、（6.250±1.323）%、（64.671±15.588）%]比较,差异无统计学意义（t值分别为0.652、1.137、0.907,P均＞0.05）.同种混合淋巴细胞反应,随着淋巴细胞的增多,增殖能力下降.磁珠分选法中,DCs与淋巴细胞的比例为1：50、1：100时,细胞增殖能力（1.502±0.055、1.507±0.029）较1：10时（1.859±0.049）降低（P均＜0.05）;贴壁培养法中,DCs与淋巴细胞的比例为1：100时,细胞增殖能力（1.545±0.066）较1：10时（2.015±0.301）降低（P＜0.05）.在DCs与淋巴细胞的比例相同时,两种方法得到的DCs在刺激T淋巴细胞方面的能力相近（P＞0.05）.结论 与磁珠分选法比较,贴壁培养2 h后的人外周血PBMC再行诱导可获得形态与功能较优的DCs,且此法稳定、简便、经济,是一种适于基础、临床研究的DCs体外培养方法.

Objective To establish a economic and stable method to induce and culture dendritic cells （DCs） from peripheral blood of human being, and compare with the magnetic activated cell sorting. Methods Monocytes were isolated from health donors peripheral blood mononuclear cells（PBMC） by density gradient separation,cultured and compared with that of cells isolated by the magnetic activated cell sorting or adherent culture,respectively. PBMC were cultured with recombinant human granulocyte macrophage colony stimulating factor （rhGM-CSF） and recombinant human interleukin-4（rhIL-4） for 6 days to induce the growth of DCs. Morphological changes was observed under inverted microscope. Meanwhile, cell viability was tested at the 3rd, 5th, 6th day,respectively. The phenotypes, like CD14, CDla, HLA-DR were analyzed with flow cytometry after PBMC were adherent cultured for 1, 2, 5 h. After adding human recombinant cytokines, the phenotypes of acquired cells surface markers, CD14, CD1a, CD86, CD83 and HLA-DR would be detected and compared with flow cytometry. T cells proliferating activity was determined by allogeneic mixed lymphocyte reaction in vitro. Results After adherent culture for 2 h, the acquired DCs showed typical morphology. Cell viability was decreased at days 5th, 6th[（53.333 ±5.774）%,（38.333 ± 7.638）%] than that at day of 3rd[（68.667 ± 3.215）%, all P 〈 0.05] with the magnetic activated cell sorting, but with adherent culture method, the difference was not statistically significant （F = 0.737,P〉 0.05） at days of 3rd, 5th, 6th[（92.667 ± 3.055）%,（94.000 ± 1.000）%,（94.667 ± 1.528）%]. Moreover,compared with the magnetic activated cell sorting, there were differences in cell viability of adherent culture method at days of 3rd, 5th, 6th（t = 9.374, 12.021,12.527, all P 〈 0.05）. Before and after using the magnetic activated cell sorting, the expression of CD14 were （32.457 ± 12.351） %, （41.914 ± 14.858）%, respectively. The difference was not statistically significant（t = 1.295, P 〉 0.05）. After culturing for 2 h, the expression of CD14[（35.267 ± 4.658）%]was higher than those of culturing for 1, 5 h[（15.033 ± 6.189）%, （21.233 ± 4.895）%, all P 〈 0.05]. Compared with the 1st day[（32.328 ± 14.517）%], the CD14 expression level[（2.200 ± 1.356）%] on surface of DCs was significantly reduced（t = 5.467, P 〈 0.05） at the 6th day of culturing, the CD1a expression level[（43.371 ±16.250）%] was remarkablely increased than that of the 1st day[（12.300 ± 6.223）%, t = 2.545, P 〈 0.05];while the expressions of CD86, CD83, HLA-DR[（16.857 ± 5.686）%,（9.343 ± 5.230）%,（72.800 ± 17.881）%] were similar（t = 0.652,1.137,0.907, all P 〉 0.05） compared with that of the 1st day[（12.550 ± 16.758）%, （6.250 ±1.323）%, （64.671 ± 15.588）%]. In mixed lymphocytes reactions, with increasing of lymphocytes, T lymphocytes proliferating activities were reduced. In the magnetic activated cell sorting, when the ratio of DCs and lymphocytes were 1： 50, 1： 100, cells proliferation ability（1.502 ± 0.055,1.507 ± 0.029） were lower than that of ratio of 1： 10（1.859 ± 0.049, all P 〈 0.05）;in adherent culture method, the ratio of DCs and lymphocytes was 1： 100, the cells proliferation ability（1.545 ± 0.066） was decreased than that of ratio 1： 10（2.015 ± 0.301, P 〈 0.05）. When the proportion of DCs and lymphocytes remained the same, the capacity to stimulate T lymphocyte was similar of the two methods（P 〉 0.05）. Conclusions Comparied with the magnetic activated cell sorting, after culture of PBMC for 2 h the induction of DCs can produce better formed and functional cells, and this method is stable, simple,economic, and is a suitable method for basic and clinical research of DCs in vitro.