胎盘血细胞因子诱导的杀伤细胞培养方法及其对肿瘤细胞杀伤效应研究  

Culture method of cytokine induced-killer cells derived from placental blood and its anticancer effect

作　　者：晁旭[1] 李宏[1] 王国全 李媛媛[1] 王斌[2] 吴洁琼[1] 赵家荣[3] 李艳君 CHAO Xu;LI Hong;WANG Guoquan;LI Yuanyuan;WANG Bin;WU Jieqiong;ZHAO Jiarong;LI Yanjun(Department of Translational Medicine,the Second Affiliated Hospital of Shaanxi University of Chinese Medicine,Xianyang 712000,China)

机构地区：[1]陕西中医药大学第二附属医院转化医学科,陕西咸阳712000 [2]陕西中医药大学药学院,陕西咸阳712046 [3]陕西中医药大学基础医学院,陕西咸阳712046

出　　处：《中华实用诊断与治疗杂志》2018年第10期958-961,共4页Journal of Chinese Practical Diagnosis and Therapy

基　　金：陕西省教育厅专项科研计划项目(16JK1207);陕西省中医药管理局科研项目(JCPT038)

摘　　要：目的建立胎盘血来源的细胞因子诱导的杀伤细胞(cytokine-induced killer,CIK)的培养方法,探讨其对肿瘤细胞的杀伤效应。方法分别采集10例健康足月产胎儿的胎盘脐静脉血(胎盘脐静脉血组)和10例健康成人的外周血(成人外周血组),以Ficoll离心法分离单个核细胞,通过定时定量加入干扰素-γ、白细胞介素-2、白细胞介素-1、CD3单克隆抗体等细胞因子,将胎盘血来源单个核细胞及外周血单个核细胞诱导培养成为CIK细胞。采用流式细胞仪检测细胞的免疫表型;用CCK-8法检测CIK细胞对人肺腺癌A549细胞、宫颈癌HeLa细胞以及肝癌HepG_2细胞的杀伤效果。结果胎盘脐静脉血组CIK细胞在培养的第6天开始呈对数增殖,第15天达增殖高峰,增殖倍数(207.91±20.08)高于成人外周血组的CIK细胞增殖倍数(169.08±23.86)(P<0.05);胎盘脐静脉血组CD3^+CD8^+、CD3^+CD56^+表达[(72.40±2.29)%、(36.63±2.53)%]均高于成人外周血组[(67.83±3.07)%、(30.96±2.89)%](P<0.05);2种来源的CIK细胞对A549、HeLa和HepG2细胞均有杀伤作用,且呈浓度依赖效应;相同效靶比下,胎盘脐静脉血组CIK细胞对肿瘤细胞杀伤作用强于成人外周血组CIK细胞。结论细胞因子可成功诱导出CIK细胞,第15天达增殖高峰;CIK细胞对肿瘤细胞有明显杀伤作用,且胎盘脐静脉血来源的CIK细胞作用更强。Objective To establish the culture method of cytokine induced-killer cells（CIK）derived from placental blood and its killing effect on cancer cells.Methods Placental umbilical vein blood from 10 healthy term fetus（fetus group）and peripheral blood of 10 healthy adults（adult group）were collected.Mononuclear cells were isolated by Ficoll centrifugation.CIK cells derived from placental umbilical vein blood and peripheral blood were induced by interferon-γ（INF-γ）,interleukin（IL）-1,IL-2,CD3 monoclonal antibodies and other cytokines.The immunophenotypes of cultural CIK cells were detected by flow cytometry.The killing effect of CIK cells on human lung adenocarcinoma A549 cells,cervical cancer HeLa cells and human hepatocellular HepG2 cells were detected by CCK-8 method.Results CIK cells in fetus group began to proliferate logarithmically on the 6th day and reached peak on the 15th day,in a multiple of 207.91±20.08,which was significantly higher than that of CIK cells in adult group（169.08±23.86）（P〈0.05）.The expressions of CD3^＋CD8^＋ and CD3^＋CD56^＋ were significantly higher in fetus group（（72.40±2.29）%,（36.63±2.53）%）than those in adult group（（67.83±3.07）%,（30.96±2.89）%）（P〈0.05）.The CIK cells in both two groups showed obvious killing effect on A549,HeLa and HepG2 cells in a concentration dependent trend,and the killing effect was stronger in fetus group than in adult group at the same effector target ratio.Conclusion CIK cells can be successfully induced by cytokines,reaching the peak of proliferation on the 15th day.CIK cells have a remarkable killing effect on cancer cells,which is stronger in placental umbilical vein blood than that in peripheral blood.

关 键 词：胎盘 肿瘤细胞 细胞因子诱导的杀伤细胞 杀伤效应 

分 类 号：R730.51[医药卫生—肿瘤]