贵州医科大学学报

2017, v.42;No.200(05) 497-502

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RTCA技术检测NK细胞联合西妥昔单抗对结直肠癌细胞的杀伤效应
The Study of Cytotoxicity Effect of NK Cells Combined with Cetuximab against Colorectal Tumor Cells with Real-time Cell Analysis System

孔娟;龙世棋;王念雪;王代琴;刘尧翠;赵星;
KONG Juan;LONG Shiqi;WANG Nianxue;WANG Daiqing;LIU Yaocui;ZHAO Xing;Department of Immunology,Guizhou Medical University;Bioengineering Institute,Guizhou Medical University;Tissue Engineering and Stem Cell Research Center,Guizhou Medical University;

摘要(Abstract):

目的:利用实时无标记细胞分析技术(RTCA)检测NK细胞介导的抗体依赖细胞介导的细胞毒(ADCC)作用。方法:采用Ficoll密度梯度离心法分离正常外周血的单个核细胞(PBMC),PBMC在体外经K562工程细胞及细胞因子诱导活化扩增为NK细胞(14 d),采用流式细胞仪检测NK细胞表面分子及分析其纯度,采用RTCA技术及乳酸脱氢酶(LDH)释放实验检测NK细胞毒作用,RTCA检测NK细胞联合西妥昔单抗对结直肠癌细胞株杀伤作用。结果:流式细胞结果显示,经过14 d体外扩增培养后NK细胞的纯度可达86.2%;NK细胞毒作用结果显示,随着效靶比的增加(1∶1、5∶1、10∶1),LDH和RTCA检测NK细胞的杀伤作用趋势基本一致;RTCA结果显示NK细胞联合西妥昔单抗对结直肠癌细胞株杀伤作用明显高于单一的NK细胞组。结论:RTCA技术为检测NK细胞介导的ADCC作用提供了一种简便、动态及灵敏的新方法。
Objective: This work aimed to explore the use of a real-time cell analysis(RTCA) system for the assessment of cytotoxicity of NK cells combined with cetuximab against colorectal tumor cells.Methods: Peripheral blood mononuclear cells(PBMC) from healthy donors were isolated by Ficoll density gradient centrifugation. NK cells were generated from PBMCs with engineered K562 cells and several recombinant cytokines in vitro. The phenotype of NK cells was analyzed by flow cytometry. The cytotoxicity of NK cells was measured by RTCA and LDH release assay,respectively. Cytotoxicity of NK cells combined with cetuximab against colorectal tumor cell lines were determined by RTCA. Result: The purity of NK cells was 86. 2% after cultured for 14 days in vitro. With the increase of the effector to target cell ratio,the cytotoxicity of NK cells measured by RTCA showed a similar trend with LDH released assay. Cytotoxicity of NK cells against colorectal tumor cells was further increased when combined with cetuximab shown by RTCA. Conclusion: The RTCA assay represents a convenient,sensitive and dynamic new technology for the assessment of NK cells mediated ADCC.

关键词(KeyWords): 实时无标记细胞分析;自然杀伤细胞;细胞毒性;西妥昔单抗;结直肠肿瘤
real time cellular analysis;natural killer cells;cytotoxicity;cetuximab;colorectal cancer

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基金项目(Foundation): 国家自然科学基金资助项目(81360346);; 贵州省应用基础研究计划重大专项资助(黔科合J重大字2003);; 贵州省科技厅社会发展攻关项目资助[黔科合SY(2012)3094号];贵州省科技厅贵阳医学院联合基金资助项目[黔科合LG字(2011)011号]

作者(Author): 孔娟;龙世棋;王念雪;王代琴;刘尧翠;赵星;
KONG Juan;LONG Shiqi;WANG Nianxue;WANG Daiqing;LIU Yaocui;ZHAO Xing;Department of Immunology,Guizhou Medical University;Bioengineering Institute,Guizhou Medical University;Tissue Engineering and Stem Cell Research Center,Guizhou Medical University;

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