两种白细胞滤器获取单个核细胞作为实验研究的生物学功能的比较*

目的：分析单采血小板去白细胞滤器(apheresis platelet leukoreduction system chambers, LRSCs)和全血去白细胞滤器(whole blood leukoreduction filters, LRFs)所收集的白细胞替代常规人全血白细胞进行实验的相关生物学功能和表型。方法：分别采用直接法和反冲洗获得LRSCs和LRFs白细胞，经人的淋巴细胞分离液进行密度梯度离心，分离出外周血单个核细胞(peripheral blood mononuclear cells, PBMCs)。用CD3激发型抗体和细胞因子白细胞介素2(interleukin, IL-2)体外扩增培养10 d，观察细胞形态、细胞增殖、细胞凋亡，并用流式细胞术(flow cytometry, FCM)进行表型分析，对比两种滤器分离出的白细胞生物学差异。结果：(1)PBMCs镜下观察：LRSCs中细胞较纯，个体大小较均一；LRFs包含部分形态较小的血小板和形态较大的粒细胞。两者细胞活力无差异。(2)LRSCs所分离的PBMCs细胞亚群以淋巴细胞为主，单核细胞次之，含极少量的粒细胞；而LRFs淋巴细胞与粒细胞比例接近，单核细胞较少。初始状态淋巴细胞群体中各细胞亚群表型相似。(3)LRSCs-PBMCs细胞，经CD3与IL-2体外刺激后，细胞活化集落大且较均一，而LRFs-PBMCs细胞集落多但分布较散。(4)体外培养第6天、第10天两组滤器间淋巴细胞各亚群表型差异无统计学意义。(5)两种滤器血PBMCs细胞凋亡率类似，且均为早期凋亡细胞比例高于晚期凋亡细胞。(6)经体外扩增培养10 d，发现两种滤器血PBMCs细胞增殖速率相当，细胞活力等同。结论：两种滤器所得的白细胞均可用于相关的基础实验研究，且LRFs含有较多的粒细胞，这也为粒细胞的研究工作提供了便利，解决了临床用血与实验用血的需求。

Comparison of the biological functions of two leukocyte filters for obtaining mononuclear cells as experimental studies*

Objective : A comparative analysis of the biological functions and phenotypes of leukocytes by the apheresis platelet leukoreduction system chambers(LRSCs) and whole blood leukoreduction filters(LRFs) in place of conventional peripheral blood leukocytes in experimental researches. Methods : LRSCs were directly flushed with PBS, and LRFs were back-flushed with PBS to obtain the leukocytes. Peripheral blood mononuclear cells(PBMCs) were isolated by density gradient centrifugation using human LymphoprepTM(Ficoll). PBMC were incubated anti-CD3 antibody and IL-2 in vitro for 10 days, and cell morphology, proliferation, apoptosis were observed, and Flow cytometry(FCM) analysis of cell phenotypes(Day0, Day6, Day10) compared the leukocytes isolated from the two different filters for biological differences. Results : (1)The PBMCs isolated from the two filters were observed under the microscope: the LRSCs cells were relatively pure and the individual size was uniform; the LRFs cells contained some small-sized platelets and large-sized granulocytes. Both cell viability were no difference. (2)FCM was used to detect the PBMCs cell population from the two filters. The LRSCs-PBMCs were mainly lymphocytes, monocytes came next, containing a very small amount of granulocytes; but the LRFs obtained PBMCs lymphocytes and granulocytes ratio were similar, and monocytes were the least. FCM analysis of PBMCs isolated from the two filters showed that the phenotypes of lymphocyte in the initial state(Day0) were similar, and there was no statistical difference. (3)The PBMCs obtained by LRSCs were stimulated by CD3 antibody and IL-2 in vitro, there were large and uniform cells activated colonies, while the LRFs-PBMCs were more colonies, but colonies were scattered. (4)FCM was used to detect the phenotypes of lymphocytes in Day6 and Day10 in vitro, and no significant difference between the two filter groups was found. (5)The apoptosis rate of PBMCs were detected after expansion. The two kinds of filter blood cells were similar, and the proportion of early apoptotic cells was higher than that of late apoptotic cells. (6)The two groups of cells were cultured in vitro for 10 days, the proliferation rate of PBMCs of the two filters was comparable, and the cell viability was equivalent. No significant difference was observed. Conclusion : This experiment confirmed that the PBMCs isolated from two different leukocyte filters blood had similar ratios of cell proliferation, activation, phenotype with the lymphocytes, except for the proportion of lymphocytes, monocytes, and granulocyte populations, indicating that the leukocytes obtained by the two filters can be preferably used in basic experimental research. The whole blood leukocyte filter contains more granulocytes, which also facilitates the research work of granulocytes and solves different blood shortage requirement in clinical and scientific research.