CIK细胞分泌因子对肝癌干细胞凋亡的影响  

The effect of secreted cytokines from killer cells induced by cytokines on apoptosis of hepatic cancer stem cells

作　　者：杨涛[1] 张曼[1] 张文军[1] 王国碧[1] 黄德鸿[1] 唐显军[1] 王宏宇 王刚 王莉[1] 肖春燕[1] 杨建中[1] 项颖[1] 李启英[1] 

机构地区：[1]重庆市肿瘤研究所血液肿瘤/生物治疗科,重庆400030 [2]上海柯莱逊生物技术有限公司,上海201201

出　　处：《肿瘤》2015年第2期147-154,共8页Tumor

基　　金：重庆市卫生局医学科研计划重点项目(编号:2012-1-127);重庆市应用开发计划项目(编号:cstc2014yykf A110031)~~

摘　　要：目的:探讨细胞因子诱导的杀伤(cytokine-induced killer,CIK)细胞的分泌因子对人肝癌干细胞(hepatic cancer stem cells,HCSCs)凋亡的影响。方法:采用无血清悬浮细胞培养法富集人肝癌细胞Hep G2获得HCSCs;用FCM法检测HCSCs表面标志物CD90及CD133的表达情况;通过对裸鼠致瘤能力的观察,鉴定HCSCs的致瘤性。以干扰素γ(interferonγ,IFNγ)、CD3单克隆抗体和重组人白细胞介素-2(recombinant human interleukin-2,rh IL-2)诱导肝癌患者外周血单个核细胞(peripheral blood mononuclear cells,PBMCs)产生CIK细胞。采用Transwell小室将不同细胞密度的CIK细胞与HCSCs接种在同一培养体系内,分隔培养24与48h后,用FCM法检测HCSCs的凋亡情况。分别采用反转录聚合酶链反应(reverse transcription-polymerase chain reaction,RT-PCR)法和蛋白质印迹法检测凋亡相关基因caspase-3 m RNA与蛋白表达的变化。结果:富集培养获得的HCSCs表面高表达干细胞标志物CD90和CD133。裸鼠致瘤能力观察表明,该HCSCs具较强的致瘤性。凋亡检测结果提示,CIK细胞的分泌因子可诱导HCSCs的凋亡,与对照组相比,凋亡率差异有统计学意义(P<0.01)。对caspase-3 m RNA与蛋白的检测结果显示,CIK细胞的分泌因子可上调HCSCs中促凋亡相关基因m RNA及蛋白的表达。结论:CIK细胞的分泌因子可诱导HCSCs发生凋亡,这可能与上调其促凋亡相关基因caspase-3的表达有关。Objective： To investigate the effect of secreted cytokines from cytokine-induced killer （CIK） cells on apoptosis of human hepatic cancer stem cells （HCSCs）. Methods： The HCSCs were enriched from human hepatic cancer cell line HepG2 by serum- free suspension culture and sphere-forming assay. The expressions of CD90 and CD133 of HCSCs were examined by flow cytometry （FCM）. The tumorigenicity of HCSCs was detected by nude mouse transplantation tumor experiment. The CIK cells were produced from suspended peripheral blood mononuclear cells （PBMCs） induced by interferon T （IFNT）, CD3 monoclonal antibody and recombinant human interleukin-2 （rhlL-2）. The HCSCs were cultured together with CIK cells at different density in a Transwell chamber and cultured separately by the membrane of the Transwell chamber for 24 and 48 h, respectively. The apoptosis of HCSCs, which were cultured with CIK cells for 24 and 48 h, was analyzed by FCM. The expression levels of caspase-3 mRNA and protein were detected by reverse transcription-polymerase chain reaction （RT-PCR） and Western blotting, respectively. Results： The result of FCM revealed that the stem cell markers CD90 and CD133 were highly expressed on the surface of HCSCs. The nude mouse transplantation tumor experiment showed that HCSCs possessed a high ability of tumorigenicity. The apoptosis of HCSCs could be significantly induced by secreted cytokines from CIK cells as compared with that of the control group （P 〈 0.01）. The caspase-3 mRNA and protein expression levels were significantly up-regulated in HCSCs induced by secreted cytokines from CIK cells. Conclusion： The secreted cytokines from CIK cells can induce apoptosis of HCSCs, and this effect may be related to upregulation of caspase-3 expression.

关 键 词：癌 肝细胞 肿瘤干细胞测定 杀伤细胞 细胞凋亡 

分 类 号：R735.7[医药卫生—肿瘤]