CIK细胞培养基及高效杀伤肝癌细胞效靶比的选择
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摘要
目的选择高效、稳定的细胞因子诱导杀伤细胞(CIK细胞)培养基及杀伤肝癌细胞的效靶比。方法采用RPMI1640、GT-T551两种培养基对5例健康人外周血单个核细胞进行体外常规诱导培养,两种培养基中分别加入0、200、500 IU/mL的IL-2,采用光学显微镜观察细胞形态变化,台盼蓝染色计数并绘制生长曲线,计算细胞扩增倍数。将CIK与肝癌7721细胞共培养,调整效靶比为10∶1、20∶1、40∶1、80∶1,72 h后进行CCK8检测,酶标仪测OD450值,计算杀伤率。结果与1640培养基比较,GT-T551培养基组各浓度IL-2下细胞扩增倍数增加(P均<0.05)。与效靶比80∶1时比较,效靶比为10∶1、20∶1、40∶1时CIK的杀伤率低(P均<0.05)。结论 GT-T551培养基加500 IU/mL IL-2的培养体系更有利于CIK细胞的增殖,CIK细胞与7721细胞的效靶比为80∶1时杀伤效应更强。
Objective The efficient and stable cytokine induced killer cell(CIK) culture medium and the effective target ratio of killing hepatocellular carcinoma cells were selected.Methods The peripheral blood mononuclear cells(PBMCs) from 5 healthy people were cultured in vitro by using RPMI1640 and GT-T551,and IL-2(0,200,and 500 IU/mL)was added to the two media,respectively.The morphology of the cells was observed by the optical microscope,and Trypan blue staining was used to count and draw the growth curve,and we calculated the cell amplification multiple.CIK was cocultured with liver cancer 7721 cell line,and the effector-target ratio was adjusted to 10∶1,20∶1,40∶1,and 80∶1.After72 h,we did the CCK8 test and measured OD450 by enzyme labeling instrument to calculate the killing rate.Results Compared with 1640 medium,the cell amplification multiples in the GT-T551 medium group at various concentrations of IL-2 increased(all P<0.05).Compared with the effector-target ratio of 80:1,the killing rate of CIK was low when the effector-target ratio was 10∶1,20∶1 and 40:1(all P<0.05).Conclusions The culture system of GT-T551 medium with 500 IU/mL IL-2 is more conducive to the proliferation of CIK cells.The cytotoxic effect reaches the peak when the effector-target ratio of CIK cells and 7721 cells is 80:1.
Objective The efficient and stable cytokine induced killer cell(CIK) culture medium and the effective target ratio of killing hepatocellular carcinoma cells were selected.Methods The peripheral blood mononuclear cells(PBMCs) from 5 healthy people were cultured in vitro by using RPMI1640 and GT-T551,and IL-2(0,200,and 500 IU/mL)was added to the two media,respectively.The morphology of the cells was observed by the optical microscope,and Trypan blue staining was used to count and draw the growth curve,and we calculated the cell amplification multiple.CIK was cocultured with liver cancer 7721 cell line,and the effector-target ratio was adjusted to 10∶1,20∶1,40∶1,and 80∶1.After72 h,we did the CCK8 test and measured OD450 by enzyme labeling instrument to calculate the killing rate.Results Compared with 1640 medium,the cell amplification multiples in the GT-T551 medium group at various concentrations of IL-2 increased(all P<0.05).Compared with the effector-target ratio of 80:1,the killing rate of CIK was low when the effector-target ratio was 10∶1,20∶1 and 40:1(all P<0.05).Conclusions The culture system of GT-T551 medium with 500 IU/mL IL-2 is more conducive to the proliferation of CIK cells.The cytotoxic effect reaches the peak when the effector-target ratio of CIK cells and 7721 cells is 80:1.
引文
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[14]杨涛,张曼,张文军,等.CIK细胞分泌因子对肝癌干细胞凋亡的影响[J].肿瘤,2015,35(2):147-154.
[15]赵花,刘征丽,许霞.宫颈癌术后输注细胞因子诱导的杀伤细胞治疗前后外周血免疫指标的变化[J].武警医学,2017,28(4):358-360,363.
[16]Schmidt J,Eisold S,Büchler MW,et al.Dendritic cells reduce number and function of CD4+CD25+cells in cytokine-induced killer cells derived from patients with pancreatic carcinoma[J].Cancer Immunol Immunother,2004,53(11):1018-1026.
[17]Lin T,Song C,Chuo DY,et al.Clinical effects of autologous dendritic cells combined with cytokine-induced killer cells followed by chemotherapy in treating patients with advanced colorectal cancer:a prospective study[J].Tumour Biol,2016,37(4):4367-4372.
[18]Huang J,Kan Q.Chemotherapy in combination with cytokine-induced killer cell transfusion:an effective therapeutic option for patients with extensive stage small cell lung cancer[J].International immunopharmacology,2017,46:170-177.
[19]周美玉,许国发,王泽新,等.细胞因子诱导的杀伤细胞联合化疗对肺癌患者免疫功能及B7-H4蛋白表达的影响[J].山西医药杂志,2017,46(2):185-188.
[20]赵群,李勇,张辉,等.奥沙利铂结合CIK细胞对人胃癌裸鼠腹膜移植模型体内抗肿瘤作用的实验研究[J].免疫学杂志,2007,14(2):212-215.
[21]徐全晓,常占国,张晓冬,等.CIK细胞联合多西他赛、奥沙利铂、替吉奥治疗晚期胃癌的疗效观察[J].中国肿瘤生物治疗杂志,2015,22(3):381-384.
[22]Kim YJ,Lim JS,Kang JS,et al.Adoptive immunotherapy of human gastric cancer with ex vivo expanded T cells[J].Arch Pharm Res,2010,33(11):1789-1795.
[23]王亚军,袁越,柳海燕,等.CIK细胞对卵巢癌化疗耐药的逆转作用及对ERCC表达的影响[J].重庆医学,2018,47(20):2649-2653.
[2]张剑军.胰腺癌免疫治疗进展[J].国际肿瘤学杂志,2007,34(1):58-61.
[3]Jin C,Li J,Wang Y,et al.Impact of cellular immune function on prognosis of lung cancer patients after cytokine-induced killer cell therapy[J].Asian Pac J Cancer Prev,2014,15(15):6009-6014.
[4]孙洁,王星,王翠芳,等.CIK细胞治疗消化系统肿瘤的效果[J].中国当代医药,2018,25(5):86-88.
[5]Rutella S,Iudicone P,Bonanno G,et al.Adoptive immunotherapy with cytokine-induced killer cells generated with a new good manufacturing practice-grade protocol[J].Cytotherapy,2012,14(7):841-850.
[6]姚惟琦,饶巍,周宏灏.嵌合抗原受体修饰CIK细胞抗肿瘤研究进展[J].中国肿瘤生物治疗杂志,2016,23(5):720-726.
[7]Yao W,Jian B,Han RD,et al.CIK cells from recurrent or refractory AML patients can be efficiently expanded in vitro and used for reduction of leukemic blasts in vivo[J].Exp Hematol,2013,41(3):241-252.
[8]李桉琪,祁元明,周哲骏,等.不同浓度IL-2对体外诱导肽特异性细胞毒性T淋巴细胞培养体系的影响[J].中国癌症杂志,2016,26(9):756-762.
[9]魏矿荣,彭侠彪,梁智恒,等.全球肝癌流行概况[J].中国肿瘤,2015,24(8):621-630.
[10]吕桂帅,陈磊,王红阳.我国肝癌研究的现状与前景[J].生命科学,2015,27(3):237-248.
[11]刘双勇,吴德全.肝癌术后复发转移原因及治疗现状[J].疑难病杂志,2018,17(3):311-314.
[12]Li QY,Shi Y,Huang DH,et al.Cytokine-induced killer cells combined with dendritic cells inhibited liver cancer cells[J].Int JClin Exp Med,2015,8(4):5601-5610.
[13]裴春颖,邢淑贤,李淑艳,等.CIK细胞体外杀伤人肝癌细胞免疫学机制的实验研究[J].哈尔滨医科大学学报,2004(2):132-134.
[14]杨涛,张曼,张文军,等.CIK细胞分泌因子对肝癌干细胞凋亡的影响[J].肿瘤,2015,35(2):147-154.
[15]赵花,刘征丽,许霞.宫颈癌术后输注细胞因子诱导的杀伤细胞治疗前后外周血免疫指标的变化[J].武警医学,2017,28(4):358-360,363.
[16]Schmidt J,Eisold S,Büchler MW,et al.Dendritic cells reduce number and function of CD4+CD25+cells in cytokine-induced killer cells derived from patients with pancreatic carcinoma[J].Cancer Immunol Immunother,2004,53(11):1018-1026.
[17]Lin T,Song C,Chuo DY,et al.Clinical effects of autologous dendritic cells combined with cytokine-induced killer cells followed by chemotherapy in treating patients with advanced colorectal cancer:a prospective study[J].Tumour Biol,2016,37(4):4367-4372.
[18]Huang J,Kan Q.Chemotherapy in combination with cytokine-induced killer cell transfusion:an effective therapeutic option for patients with extensive stage small cell lung cancer[J].International immunopharmacology,2017,46:170-177.
[19]周美玉,许国发,王泽新,等.细胞因子诱导的杀伤细胞联合化疗对肺癌患者免疫功能及B7-H4蛋白表达的影响[J].山西医药杂志,2017,46(2):185-188.
[20]赵群,李勇,张辉,等.奥沙利铂结合CIK细胞对人胃癌裸鼠腹膜移植模型体内抗肿瘤作用的实验研究[J].免疫学杂志,2007,14(2):212-215.
[21]徐全晓,常占国,张晓冬,等.CIK细胞联合多西他赛、奥沙利铂、替吉奥治疗晚期胃癌的疗效观察[J].中国肿瘤生物治疗杂志,2015,22(3):381-384.
[22]Kim YJ,Lim JS,Kang JS,et al.Adoptive immunotherapy of human gastric cancer with ex vivo expanded T cells[J].Arch Pharm Res,2010,33(11):1789-1795.
[23]王亚军,袁越,柳海燕,等.CIK细胞对卵巢癌化疗耐药的逆转作用及对ERCC表达的影响[J].重庆医学,2018,47(20):2649-2653.