不同培养基与血清的培养体系对细胞因子诱导杀伤细胞增殖和功能的影响  

Effects of different cell culture medium and different sources of serum on proliferative and functional activities of cytokine-induced killer cells

作　　者：高群[1,2] 王菲[1,3] 王丽萍[2] 杨黎[1] 张震[1,2] 岳冬丽[1,2] 王盟[1,4] 张毅[1,2,3,5] 

机构地区：[1]郑州大学第一附属医院生物细胞治疗中心,河南郑州450052 [2]郑州大学第一附属医院肿瘤科,河南郑州450052 [3]郑州大学生命科学院,河南郑州450001 [4]郑州大学第一附属医院消化内科,河南郑州450052 [5]河南省高等学校临床医学重点学科开放实验室,河南郑州450052

出　　处：《中国肿瘤生物治疗杂志》2014年第6期680-686,共7页Chinese Journal of Cancer Biotherapy

基　　金：国家自然科学基金资助项目(No.81171986;No.81271815);卫生部科研攻关基金资助(No.20110110001);河南省科技厅基础与前沿技术研究基金资助(No.112300410153;No.122300410155);河南省科技厅科技创新人才计划资助(No.124200510006);郑州大学第一附属医院院内创新团队基金资助~~

摘　　要：目的:探讨不同细胞培养基及血清的培养体系对细胞因子诱导杀伤(cytokine-induced killer,CIK)细胞的增殖和功能的影响。方法:采集6例肺癌患者外周血,分离获得单个核细胞,按照不同血清与培养基分为8组:GT-T551培养基+患者自体血清组、GT-T551培养基+健康人血清组、GT-T551培养基+FBS组、GT-T551培养基组、RPMI 1640培养基+患者自体血清组、RPMI 1640培养基+健康人血清组、RPMI 1640培养基+FBS组及RPMI 1640培养基组。采用CFSE染色法检测细胞增殖能力,流式细胞术检测CIK细胞CD3+CD8+T细胞及CD3+CD4+T细胞颗粒酶B、IFN-γ、穿孔素的分泌情况及以白血病NB4、K562细胞为靶细胞时CD3+CD56+T细胞、CD3+CD4+T细胞及CD3+CD8+T细胞表面CD107a的表达。结果:GTT551培养基加入患者自体血清组CIK细胞的增殖指数显著高于其他各组(P<0.05)。GT-T551培养基或RPMI 1640培养基中加入患者自体血清组CD4+T细胞颗粒酶B的分泌水平均显著高于加健康人血清组[(22.85±3.50)%vs(13.28±1.75)%,(22.57±3.45)%vs(15.37±4.08)%,均P<0.01],而且GT-T551+患者自体血清组CD8+T细胞分泌IFN-γ的能力显著高于加健康人血清组(P<0.05)。以白血病细胞系NB4和K562细胞作为靶细胞时,检测CD3+CD56+NKT细胞表面CD107a的表达,GT-T551培养基中加入患者自体血清组优于加健康人血清组[(7.10±1.94)%vs(2.73±0.79)%,(8.00±1.82)%vs(3.03±0.78)%,P<0.01],同时优于RPMI1640+患者自体血清组[(4.45±1.96)%、(3.30±1.47)%,P<0.01]。结论:GT-T551培养基加患者自体血清的培养体系更有利于CIK细胞的增殖、细胞因子分泌及发挥杀伤功能,可推荐作为CIK细胞最佳培养体系。Objective： To investigate the effects of different culture media and different sources of serum on proliferativeand functional activities of cytokine-induced killer （CIK） cells. Methods： Mononuclear cells were isolated from peripher- al blood of 6 lung cancer patients and were induced to differentiate into CIK cells in 8 different culture conditions ： GT- T551 ＋ autologous serum, GT-T551 ＋ serum from a healthy individual, GT-T551 ＋ FBS, GT-T551, RPMI 1640 ＋ au- tologous, RPMI 1640 ＋ serum from a healthy individual, RPMI 1640 ＋ FBS, and RPMI 1640. After induction for 6 days, cell proliferation was assessed carboxyfluoreseein diacetate succinimidyl sster （CFSE） labeling assay, and CD3＋ CD8 ＋ cell proportion, CD3＋ CD4＋ T cell expression of Granzyme B and IFN-y/and perforin and the expression of CDIO7a on CD3 ＋ CD56＋ , CD3 ＋ CD4 ＋ and CD3 ＋ CD8 ＋ T cells in the presence of leukemia NB4 and K562 cells as targets were as- sessed by flow cytometry. Results： The proliferation index of CIK cells cultured in GT-T551 medium supplemented with autologous serum was significantly higher than that in other culture conditions （ P 〈 0.05 ）. The expression of Granzyme B on CD4 ＋ T cells was significantly higher （P 〈 0.01 ） in the GT-T551 ＋ autologous serum group [ 22.85 ±3.50 ] % than in the GT-T551 ＋ healthy serum group [ 13.28± 1.75 ] % and significantly higher （ P 〈 0.01 ） in the RPMI 1640 ＋ autolo- gous serum group [22.57 ± 3.45 ] % than in the RPMI 1640 ＋ healthy serum group[ 15.37 ±4.08 ] %. Furthermore, the amount of IFN-y,/secreted from CD8 ~ T cells was significantly higher in the GT-T551 ＋ autologous serum groups compared to GT-T551 ＋ healthy serum group. In the presence of killing NIM and K562 ceils, the expression of CD107a on the CD3 ＋ CD56＋ NKT cells was significantly higher in the GT-T551 ＋ autologous serum group （ [ 7.10± 1.94 ] %, [ 8.00 ± 1.821%） than in the GT-T551 ＋healthy serum group （[2.73 ±0.79]%, 13.03 ±0.78

关 键 词：细胞因子诱导的杀伤细胞 培养基 血清 增殖 细胞毒性 白血病细胞 

分 类 号：R735.1[医药卫生—肿瘤] R392.12[医药卫生—临床医学]