贵州医科大学学报

2021, v.46;No.244(01) 1-8+15

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溶瘤呼肠孤病毒联合CD30/CD16A双特异性抗体对NK细胞介导的ADCC效应的影响
Effect of oncolytic reovirus combined with CD30/CD16A bispecific antibodies on NK cells-mediated ADCC

廖春香;龙世棋;安媛媛;车启元;王念雪;陈亮;杨薇;赵星;
LIAO Chunxiang;LONG Shiqi;AN Yuanyuan;CHE Qiyuan;WANG Nianxue;CHEN Liang;YANG Wei;ZHAO Xing;Stem Cell and Tissue Engineering Research Center,Guizhou Medical University;Department of Immunology,Guizhou Medical University,School of Basic Medicine;Department of Thoracic Breast Surgery,Anshun People's Hospital;Department of Oncology,Clinical Medical College,Guizhou Medical University;

摘要(Abstract):

目的探究溶瘤呼肠孤病毒(reovirus)联合CD30/CD16A双特异性抗体(BsAbs)对自然杀伤细胞(NK)活性及抗体依赖的细胞介导的细胞毒作用(ADCC)的影响。方法采集健康献血者肝素抗凝静脉血30 m L,分离外周血单个核细胞(PBMC),采用免疫磁珠筛选获得NK细胞,采用流式细胞术检测NK细胞纯度;溶瘤呼肠孤病毒(reovirus)预处理NK细胞(Reo-NK组),以单独NK组为对照,采用乳酸脱氢酶(LDH)释放实验检测2组NK细胞的活化作用; reovirus联合(CD30/CD16A) BsAb杀伤KARPAS-299细胞,分为IgG1处理组(IgG1+NK组)、CD30/CD16A处理组(CD30/CD16A+NK组)、病毒联合IgG1处理组(IgG1+Reo-NK组)及病毒联合CD30/CD16A处理组(CD30/CD16A+Reo-NK组),采用LDH释放实验检测各组NK细胞的活化作用,采用流式细胞术检测各组NK细胞表面活化标注物CD69和细胞毒性颗粒CD107a的表达;用reovirus联合(CD30/CD16A) BsAb处理NK细胞(Reo-NK+CD30/CD16A组),以NK联合(CD30/CD16A) BsAb为对照(NK+CD30/CD16A组),采用LDH释放试验检测2组NK细胞对Raji细胞的杀伤效应。结果体外新鲜分离的NK细胞纯度可达70%以上;与单独NK细胞组相比,Reo-NK组DLD-1细胞的杀伤率明显增高(P <0.01); KARPAS-299细胞上CD30分子的阳性率高达90%以上; IgG1与(CD30/CD16A) BsAb抗体浓度大于1.00×10-12mol/L时,与NK+IgG1组相比,NK+CD30/CD16A组促进KARPAS-299细胞死亡率(P <0.05);与Reo-NK相比,Reo-NK+CD30/CD16A组能明显促进KARPAS-299细胞死亡率(P <0.01);随着reovirus滴度增加,NK细胞表面活化标致物CD69及细胞毒性物质CD107a阳性率随之上升; reovirus与(CD30/CD16A) BsAbs相比可以进一步促进NK细胞的活化。结论 Reovirus可促进NK细胞的活化并增强其细胞毒作用,联合(CD30/CD16A) BsAbs后可进一步促进NK细胞介导的ADCC效应。
Objective To explore the effect of oncolytic reovirus combined with CD30/CD16 A bispecific antibodies( BsAbs) on natural killer( NK) cell activity and antibody-dependent cellmediated cytotoxicity( ADCC). Methods Thirty milliliter of heparin anticoagulated venous blood from healthy blood donors were collected. Peripheral blood mononuclear cells( PBMC) were isolated.NK cells were extracted by immunomagnetic beads from PBMC. NK cell purity was examined by flow cytometry. NK cells were pretreated with oncolytic reovirus as( Reo-NK group),and untreated NK cells as control group. Lactate dehydrogenase( LDH) release was used to examine the activation of NK cells. Reovirus combined with( CD30/CD16 A) BsAb in killing KARPAS-299 cell experiment included IgG1 + NK group,CD30/CD16 A + NK group,IgG1 + Reo-NK group,CD30/CD16 A + Reo-NK group. LDH release assay was used to detect the activation of NK cells in each group. Flow cytometry was used to detect the expression of CD69 and CD107 a on the surface of NK cells. NK cells were treated with reovirus combined with( CD30/CD16 A) BsAbs( Reo-NK + CD30/CD16 A),and NK combined with( CD30/CD16 A) BsAbs as control( NK + CD30/CD16 A). LDH release assay was used to detect the killing effect of NK cells on Raji cells. Results The purity of freshly isolated NK cells in vitro was over 70%. Compared with the NK cell group alone,the killing rates of NK cells on DLD-1 cells in the Reo-NK group was significantly increased( P < 0. 01),and CD30+KARPAS-299 cells were 90%. When the concentration of IgG1 and( CD30/CD16 A) BsAb antibodies is greater than1. 00 × 10-12 mol/L,NK + CD30/CD16 A group promotes the death rate of KARPAS-299 cells( P <0. 05) when compared with the NK + IgG1 group. Relative to Reo-NK,Reo-NK + CD30/CD16 A group can significantly promote KARPAS-299 cell mortality( P < 0. 01). As the titer of reovirus increases,activation marker CD69 of NK cells and the positive rate of cytotoxic substance CD107 a increases accordingly. Reovirus could promote the killing rate of NK cells to Raji cells,but( CD30/CD16 A)BsAbs could not. Conclusion Reovirus can promote the activation of NK cells and enhance its cytotoxicity,and the combination of( CD30/CD16 A) BsAbs can further promote the NK cell-mediated ADCC.

关键词(KeyWords): 抗体,双特异性;呼肠孤病毒;自然杀伤细胞;抗体依赖的细胞介导的细胞毒作用;溶瘤作用;细胞毒效应
antibody,bispecific(BsAbs);reovirus;natural killer(NK) cell;antibody-dependent cell-mediated cytotoxicity(ADCC);oncolysis;cytotoxic effect

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金(81860542);; 贵州省普通高等学校科技拔尖人才支持计划[黔科合KY字(2018)049];; 贵州省优秀青年科技人才项目[黔科合平台人才(2019)5663]

作者(Author): 廖春香;龙世棋;安媛媛;车启元;王念雪;陈亮;杨薇;赵星;
LIAO Chunxiang;LONG Shiqi;AN Yuanyuan;CHE Qiyuan;WANG Nianxue;CHEN Liang;YANG Wei;ZHAO Xing;Stem Cell and Tissue Engineering Research Center,Guizhou Medical University;Department of Immunology,Guizhou Medical University,School of Basic Medicine;Department of Thoracic Breast Surgery,Anshun People's Hospital;Department of Oncology,Clinical Medical College,Guizhou Medical University;

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DOI: 10.19367/j.cnki.2096-8388.2021.01.001

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