不同刺激因子对人CIK细胞功能影响  

Effects of Different Stimulatory Factors on Functions of CIK Cells

作　　者：刘军权[1] 朱云[1] 陈复兴[1] 周燏[1] 姬会春[1] 杨宛莹[1] 吕小婷[1] 张颂[1] 陶征中[1] 李昳[1] 

机构地区：[1]中国人民解放军第九七医院肿瘤生物治疗中心,江苏徐州221004

出　　处：《中国实验血液学杂志》2013年第4期1021-1026,共6页Journal of Experimental Hematology

基　　金：南京军区医学科技创新课题(11MA040)

摘　　要：本研究观察不同的细胞刺激因子共刺激对人CIK细胞增殖和功能的影响。用淋巴细胞分离液分离人外周血单个核细胞(PBMNC),按常规方法从PBMNC培养细胞因子诱导的杀伤细胞(CIK细胞),然后根据加入CD28 mAb、IL-15和IL-21将实验分为5组:对照组(CIK),CB28+IL-15+IL-21组,IL-15+IL-21组,CD28+IL-15组和CD28+IL-21组。用全自动五分类血液分析仪计数CIK细胞的增殖能力;用流式细胞术测定细胞刺激因子诱导的CIK细胞的粒酶B(granzyme B),穿孔蛋白(perforin)和CD107α等分子的变化;用ELISA法检测细胞因子IL-10、IL-12、INF-γ和TNF-α的含量;乳酸脱氢酶释放法测定细胞刺激因子共刺激的细胞对人肺癌细胞株A549(A549)、乳腺癌细胞株MFC-7(MFC-7)和人黑素瘤细胞株HME1(HME1)的杀伤活性。结果表明,在CIK细胞培养体系中加入不同的刺激因子,细胞增殖能力有明显的差异,以含CD28、IL-15和IL-21组细胞增殖倍数最高,在培养第10日时该组的增殖倍数为255.3±6.3,明显高于对照组,IL-21+IL-15组和CD28+IL-21组细胞增殖倍数分别为166.6±13.5、199.4±15.0和228.8±16.6(P<0.05),添加CD28和IL-15组则穿孔蛋白含量明显高于其他组。所有共刺激组的穿孔蛋白、粒酶B和CD107a表达百分率均明显高于对照组(P<0.05)。对A549、MFC-7和HME1细胞杀伤活性以含CD28+IL-15组最高(82.2%、59.3%和70.6%),明显高于对照组(60.9%、49.6%和48.4%)(P<0.05)。在CIK细胞培养体系中增加CD28+IL-15+IL-21组中细胞分泌IFN-γ量显著高于其他各组(P<0.05)。结论:不同刺激因子活化的CIK细胞的增殖能力、分泌细胞因子和杀伤活性有明显差异,在培养体系中增加相应的细胞刺激因子对细胞功能定向培养有一定意义。This study was aimed to inuestigate the effects of different stimulatory facrors on proliferation and function of cytokine induced killer（CIK） cells.Peripheral blood mononuclear cells（PBMNC） were separated by Ficoll-Hypacue gradient.According to supplement of different stimulatory factors（CD28 mAb,IL-15 and IL-21）,the experiment was divided into five groups：contod group（CIK）,CB28＋IL-15＋IL-21 group,IL-15＋IL-21 group,CD28＋IL-15 group and CD28＋IL-21 group.Effects of different stimulatory facros on the proliferation of CIK cells were assayed by an automated hematology analyzer.Changes of granzyme B,perforin and CD107a were detected by flow cytometry.IL-10,IL-12,INF-γ and TNF-α were quantified by ELISA.Cytotoxicities on lung cancer cell line A549,breast adenocarcinoma cell line MFC-7 and human melanoma cell line HME1 were examined by lactate dehydrogenase release method.The results showed that there were significant differences among different groups.The highest proliferation index on days 10 was observed in group CD28mAb,IL-15 and IL-21（255.3±6.3）,which was higher than control group,IL-21＋IL-15 group and CD28 mAb＋IL-21group（166.6±13.5,199.4±15.0 and 228.8±16.6）（P〈0.05）.The expression of perforin in CD28 mAb＋IL-15 group was higher than the other groups.The expression of perforin,GranB and CD107a of costimulatory groups was higher than control group.The cytotoxicities of CD28 mAb＋IL-15 group on A549,MFC-7 and HME1 cells（82.2%、59.3% and 70.6%） were much higher than that of control group（60.9%、49.6% and 48.4%）（P〈0.05）.The highest IFN-γsecretion was found in CD28 mAb,IL-15 and IL-21 groups.It is conduded that there are significant difference of proliferative capacity,cytokine secretion and cytotoxicity after being activated by different stimulatory factors.Adding corresponding stimulatory factors into the culture system displays a greal value for target cells culture.

关 键 词：共刺激 CIK细胞 细胞因子 杀伤活性 

分 类 号：R979.1[医药卫生—药品]