两种培养基的培养体系对CIK细胞增殖和功能的影响
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摘要
目的观察细胞因子诱导的杀伤细胞(CIK细胞)增殖情况,检测CIK细胞的表面分子表型和体外对白血病细胞K562的杀伤作用。方法通过用H3培养基培养的分别来源于脐带血和患者自体外周血分离的单个核细胞(PBMC),添加重组人γ干扰素(IFN-γ)与CD3单抗,体外诱导CIK细胞,在培养的第7天分别加入H3培养基和T551培养基继续诱导培养至14d。计数观察CIK细胞增殖能力,流式检测细胞表面CD3、CD56的表达情况,CCK8法检测两组培养基条件下对白血病细胞K562的杀伤效果。结果动态计数及表型分析结果表明,H3及H3~+T551培养的CIK细胞扩增倍数(包括脐带血CIK总细胞数和自体CIK总细胞数)在第14天均分别达到76.9倍、62.3倍;两种培养条件下脐带血CIK细胞中CD3~+CD56~+双阳性细胞含量分别是(16.70±2.72)%和(10.80±2.59)%,自体CIK细胞中CD3~+CD56~+双阳性细胞含量分别是(11.23±6.64)%和(10.70±6.42)%;体外杀瘤实验表明,当效靶比为5∶1时,H3培养的脐带血CIK细胞杀伤率达到(33.50±9.99)%,显著高于H3~+T551培养脐带血CIK细胞的(20.3±6.76)%,差异有统计学意义(P=0.011),而H3和H3~+T551培养的自体CIK细胞杀伤率分别是(59.67±27.59)%和(42.13±19.47)%,差异无统计学意义(P=0.080)。结论 H3培养的脐带血和自体CIK细胞具有较强的体外抗白血病癌细胞活性,可应用于临床上白血病的过继性免疫治疗。
Objective To observe the proliferation ability of cytokine-induced killer(CIK)cells and to detect surface molecule phenotype of CIK cells and in vitro killing effect to leukemia K562 cells.Methods The cord blood lymphocytes and peripheral blood mononuclear cells(PBMCs)from patients were cultured by the H3 medium and added with human recombinant interferon(IFN)-γand CD3 monoclonal antibodies(mAb)for inducing CIK cells in vitro.The H3 medium and T551 medium were respectively added on 7dof culture.The induction and culture were continued until 14 d.The CIK cells proliferation ability was observed by cell count and the expressions of CD3 and CD56were detected by flow cytometry.The killing effect of CIK cells on leukemia cells was tested by CCK8 assays.Results The dynamic counting and phenotype analysis results revealed that the CIK cells amplification times(including cord blood CIK total cells and autologous CIK total cells)cultured by H3 and H3+T551reached 76.9times and62.3times on 14drespectively;the CD3+CD56+double positive cells contents of cord blood CIK cells under the two kinds of culture condition were(16.7±2.7)% and(10.8±2.6)% respectively,while which of autologous CIK cells were(11.2±6.6)% and(10.7±6.4)% respectively;the in vitro killing-tumor test revealed that when the effector to-target ratio was 5:1,the cell cytotoxic activity of cord blood CIK cells cultured by H3reached(33.50±9.99)%,which was significantly higher than(20.3±6.76)% of cord CIK cells cultured by H3+T551(P=0.011),while which of autologous CIK cells cultured by H3+T551 were(59.67±27.59)% and(42.13±19.47)% respectively,but the difference was not significant(P=0.080).Conclusion Cord blood and autologous CIK cells incubated by H3 have stronger in vitro anti-leukemia cells activity and can be used in the adoptive immunotherapy of leukemia in clinic.
Objective To observe the proliferation ability of cytokine-induced killer(CIK)cells and to detect surface molecule phenotype of CIK cells and in vitro killing effect to leukemia K562 cells.Methods The cord blood lymphocytes and peripheral blood mononuclear cells(PBMCs)from patients were cultured by the H3 medium and added with human recombinant interferon(IFN)-γand CD3 monoclonal antibodies(mAb)for inducing CIK cells in vitro.The H3 medium and T551 medium were respectively added on 7dof culture.The induction and culture were continued until 14 d.The CIK cells proliferation ability was observed by cell count and the expressions of CD3 and CD56were detected by flow cytometry.The killing effect of CIK cells on leukemia cells was tested by CCK8 assays.Results The dynamic counting and phenotype analysis results revealed that the CIK cells amplification times(including cord blood CIK total cells and autologous CIK total cells)cultured by H3 and H3+T551reached 76.9times and62.3times on 14drespectively;the CD3+CD56+double positive cells contents of cord blood CIK cells under the two kinds of culture condition were(16.7±2.7)% and(10.8±2.6)% respectively,while which of autologous CIK cells were(11.2±6.6)% and(10.7±6.4)% respectively;the in vitro killing-tumor test revealed that when the effector to-target ratio was 5:1,the cell cytotoxic activity of cord blood CIK cells cultured by H3reached(33.50±9.99)%,which was significantly higher than(20.3±6.76)% of cord CIK cells cultured by H3+T551(P=0.011),while which of autologous CIK cells cultured by H3+T551 were(59.67±27.59)% and(42.13±19.47)% respectively,but the difference was not significant(P=0.080).Conclusion Cord blood and autologous CIK cells incubated by H3 have stronger in vitro anti-leukemia cells activity and can be used in the adoptive immunotherapy of leukemia in clinic.
引文
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[13]陈娟,卢光琇.CIK细胞的生物特性和应用研究新进展[J].肿瘤药学,2014,4(3):166-171.
[14]Lu X,Zhu A,Cai X,et al.Role of NKG2Din cytokine-induced killer cells against multiple myeloma cells[J].Cancer Biol Ther,2012,13(8):623-629.
[15]Wang W,Li R,Meng M,et al.microRNA profiling of CD3+CD56+cytokine-induced killer cells[J].Sci Rep,2015(5):9571.
[2]June CH,Riddell SR,Schumacher TN,et al.Adoptive cellular therapy:a race to the finish line[J].Sci Transl Med,2015,25(7):280.
[3]Li DR,Yang YH,Gao L,et al.The possible molecular regulation mechanism of CIK cells inhibiting the proliferation of Human Lung Adenocarcinoma NCL-H157Cells[J].Open Med,2016,11(1):1-6.
[4]Jakel CE,Schmidt-Wolf IG.An update on new adoptive immunotherapy strategies for solid tumors with cytokineinduced killer cells[J].Expert Opin Biol Ther,2014,14(7):905-916.
[5]Kim JS,Park YS,Kim JY,et al.Inhibition of human pancreatic tumor growth by cytokine-induced killer cells in nude mouse xenograft model[J].Immune Netw,2012,12(6):247-252.
[6]Pan QZ,Pan K,Wang QJ.et al.Annexin A3as a potential target for immunotherapy of liver cancer stem-like cells[J].Stem Cells,2015,33(2):354-366.
[7]Jiang J,Wu C,Lu B.Cytokine-induced killer cells promote antitumor immunity[J].J Transl Med,2013(11):83.
[8]殷雪,徐鑫,赵瑶,等.抗体介导的高效CIK(AMHE-CIK)细胞体外诱导的数种优化方案的比较研究[J].中国实验血液学杂志,2016,24(1):46-48.
[9]Amadori S,Del Principe MI,Venditti A.Advances in the treatment of elderly and frail patients with acute myeloid leukemia[J].Curr Opin Oncol,2014,26(6):663-669.
[10]Loh ML,Mullighan CG.Advances in the genetics of highrisk childhood B-progenitor acute lymphoblastic leukemia and juvenile myelomonocytic leukemia:implications for therapy[J].Clin Cancer Res,2012,18(10):2754-2767.
[11]黄谋珍,白俊,李峰松,等.PHA诱导的CIK细胞的生物学特性及其对K562细胞杀伤活性影响的研究[J].中国实验血液学杂志,2014,22(1):64-68.
[12]张小锋.CIK细胞杀瘤实验前后细胞表型与细胞毒性相关性研究[J].中国医药导刊,2013(5):870-871.
[13]陈娟,卢光琇.CIK细胞的生物特性和应用研究新进展[J].肿瘤药学,2014,4(3):166-171.
[14]Lu X,Zhu A,Cai X,et al.Role of NKG2Din cytokine-induced killer cells against multiple myeloma cells[J].Cancer Biol Ther,2012,13(8):623-629.
[15]Wang W,Li R,Meng M,et al.microRNA profiling of CD3+CD56+cytokine-induced killer cells[J].Sci Rep,2015(5):9571.