分泌IL-21的白血病细胞系的建立及其抑瘤作用

Establishment of leukemia cell lines expressing IL-21 and analysis of anti-tumor function

目的建立及鉴定稳定表达白介素21的4种白血病细胞系,并初步分析其刺激活化的淋巴细胞对肿瘤细胞的杀伤功能。方法构建携带白介素21和红色荧光蛋白基因的质粒,包装慢病毒,感染4种白血病细胞系,连续传代后,通过荧光显微镜、流式细胞仪和ELISA法鉴定建立的4种细胞系。以稳定分泌IL-21白血病细胞为抗原刺激外周血单个核细胞增殖,细胞分析计数仪检测增殖倍数,流式细胞仪分析淋巴细胞的表型,细胞计数试剂盒(CCK8)检测增殖后的淋巴细胞对肿瘤细胞的杀伤作用。结果建立稳定表达IL-21的4种细胞系:K562-IL-21、THP-1-IL-21、jurkat E6-1-IL-21和RPMI8226-IL-21。3份人PBMC在4种分泌IL-21的白血病细胞的作用下都有增殖,增殖倍数在1.147±0.057~2.725±0.345倍之间。不同数量的IL-21分泌白血病细胞都可激活淋巴细胞增殖,增殖的倍数在1.127±0.152~2.213±0.200之间。增殖后CD3+T淋巴细胞和CD56+NK细胞的比例降低(P<0.05),CD19+B淋巴细胞比例增加(P<0.05);由分泌IL-21白血病细胞激活后的淋巴细胞对母系白血病细胞的杀伤作用明显高于对照组。由分泌IL-21白血病细胞激活后的淋巴细胞对母系白血病细胞和其他7种肿瘤细胞均有杀伤作用,杀伤率在(28.68±9.31)%^(78.45±0.61)%之间。结论 4种分泌IL-21的白血病细胞可刺激PBMC增殖并激活其对肿瘤细胞的杀伤作用。

Objective To establish leukemia cell lines expressing IL-21 constitutively,and to analyze the activation function on lymphocytes by those cell lines.Methods Four leukemia cell lines were infected with lentivirus carrying IL-21 and RFP genes.Fluorescent microscope was used for observing the expression of RFP and flow cytometry was used for analyzing the percentage of RFP-positive cells.The concentration of IL-21 in the medium was detected by ELISA.After stimulation with the IL-21 stable-expressing leukemia cell lines,the amplification of PBMC were calculated by cell counter and the surface markers were analyzed by flow cytometry,while their cytotocicity towards different tumor cells were assessed with CCK8 test.Results Four leukemia cell lines stable-expressing IL-21 were established,namely,K562-IL-21,THP-1-IL-21,jurkat E6-1-IL-21 and RPMI8226-IL-21.The lymphocytes amplification of 3 different samples varied from 1.147 ±0.057 to 2.725 ±0.345 times and the amplification folds of lymphocytes stimulated by different numbers of leukemia cells varied from 1.127 ±0.152 to 2.213 ±0.200.The percentage of CD3 ＋ T cell and CD56 ＋ NK cell decreased （P 〈 0.05） while CD19 ＋ B cells increased （P 〈 0.05） after stimulation.The killing rate of activated lymphocytes is much higher than that of control.The activated lymphocytes had powerful killing capacity on their stimulating leukemia cells and 7 other tumor cells and the killing rates ranged from 28.68 ± 9.31% to 78.45 ± 0.61%.Conclusions Four leukemia cell lines stable-expressing IL-21 may activate PBMC to kill different tumor cells.