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

• 1:贵州医科大学免疫学教研室
• 2:贵州医科大学生物与工程学院
• 3:贵州医科大学组织工程与干细胞实验中心

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

Abstract:

Keywords:

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

参考文献(References)：

• [1]Peruzzi G,Masilamani M,Borrego F,et al.Endocytosis as a mechanism of regulating natural killer cell function:unique endocytic and trafficking pathway for CD94/NKG2A[J].Immunol Res,2009(1-3):210-222.
• [2]陈力川,汪思亮,余苏云,等.NK细胞在肿瘤免疫治疗中的研究进展[J].肿瘤,2017(1):101-106.
• [3]Zhao X,Rajasekaran N,Chester C,et al.Reovirus activated NK cells show enhanced cetuximab mediated antibody-dependent cellular cytotoxicity against colorectal cancer cells[J].Journal for immunotherapy of cancer,2015(2):1-10.
• [4]Zhao X,Rajasekaran N,Chester C,et al.Natural killer cells activated by oncolytic reovirus enhance cetuximab mediated antibody dependent cellular cytotoxicity in an in vitro and In vivo model of colorectal cancer[J].Blood,2015(23):3439.
• [5]Zhao X,Rajasekaran N,Chester C,et al.Abstract B082:Reovirus treated NK cells exhibit enhanced cetuximab mediated antibody-dependent cellular cytotoxicity against colorectal cancer cell lines[J].Cancer Immunology Research,2016(1):B082-B082.
• [6]王志华.NK细胞抗肿瘤临床转化研究进展[J].中国肿瘤生物治疗杂志,2016(3):419-426.
• [7]Somanchi SS,Mc Culley KJ,Somanchi A,et al.A novel method for assessment of natural killer cell cytotoxicity using image cytometry[J].PLo S One,2015(10):e0141074.
• [8]刘春雨,王兰,郭玮,等.基于报告基因的抗CD20单克隆抗体ADCC生物学活性测定方法的建立[J].药学学报,2015(1):94-98.
• [9]Yamashita M,Kitano S,Aikawa H,et al.A novel method for evaluating antibody-dependent cell-mediated cytotoxicity by flowcytometry using cryopreserved human peripheral blood mononuclear cells[J].Sci Rep,2016(6):19772.
• [10]郭继强,韩亚萍,李芳,等.流式细胞术三种染色方法检测体外纯化扩增的NK细胞的细胞毒作用比较[J].中国实验血液学杂志,2016(6):1691-1697.
• [11]Teng Z,Kuang X,Wang J,et al.Real-time cell analysis-a new method for dynamic,quantitative measurement of infectious viruses and antiserum neutralizing activity[J].J Virol Methods,2013(2):364-370.
• [12]Ramis G,Martínez-Alarcón L,Quereda JJ,et al.Optimization of cytotoxicity assay by real-time,impedance-based cell analysis[J].Biomed Microdevices,2013(6):985-995.
• [13]Ozdemir A,Ark M.x CELLigence real time cell analysis system:a new method for cell proliferation and cytotoxicity[J].Niche,2013(3):15-17.
• [14]Roshan MM,Young A,Reinheimer K,et al.Dynamic assessment of cell viability,proliferation and migration using real time cell analyzer system(RTCA)[J].Cytotechnology,2015(2):379-386.
• [15]Martinez-Serra J,Gutierrez A,Muoz-CapóS,et al.x CELLigence system for real-time label-free monitoring of growth and viability of cell lines from hematological malignancies[J].Onco Targets Ther,2014(7):985-994.
• [16]Limame R,Wouters A,Pauwels B,et al.Comparative analysis of dynamic cell viability,migration and invasion assessments by novel real-time technology and classic endpoint assays[J].PLo S One,2012(10):e46536.
• [17]Kho D,Mac Donald C,Johnson R,et al.Application of x CELLigence RTCA biosensor technology for revealing the profile and window of drug responsiveness in real time[J].Biosensors(Basel),2015(2):199-222.
• [18]Sandra NG,Leslie G,Amy MN.Real time cellular analysis as a novel approach for in vitro cytotoxicity testing of medical device extracts[J].J Biomed Mater Res A,2013(101):2097-2106.
• [19]ZHANG LL,Li-Na MA,Dan YAN,et al.Dynamic monitoring of the cytotoxic effects of protoberberine alkaloids from Rhizoma Coptidis on Hep G2 cells using the x CELLigence system[J].Chinese journal of natural medicines,2014(6):428-435.
• [20]王甫厚,陈绍先,郭彩云,等.LDH释放法测NK细胞毒的方法学研究[J].中国免疫学杂志,1990(2):115-117.

文章评论(Comment)：