人CD137抗体促进NK细胞对乳腺癌细胞特异性杀伤作用的体外研究  

Effects of human CD137 antibody on specific cytotoxicity in NK cells against breast cancer cells in vitro

作　　者：刘鲁宁 陈雪梅 马恩奇 田清艳[1] 刘倩倩 刘韬 Liu Luning;Chen Xuemei;Ma Enqi;Tian Qingyan;Liu Qianqian;Liu Tao(Department of Oncology Rehabilitation,Shenzhen Luohu People's Hospital,the Third Affiliated Hospital of Shenzhen University,Shenzhen 518001,China)

机构地区：[1]深圳市罗湖区人民医院(深圳大学第三附属医院)肿瘤康复科,518001

出　　处：《中华细胞与干细胞杂志（电子版）》2020年第6期346-353,共8页Chinese Journal of Cell and Stem Cell（Electronic Edition）

基　　金：深圳市医学重点学科建设经费资助(SZXK062);深圳市科技创新项目(JCYJ20170307171034705、JCYJ20170412155231633)。

摘　　要：目的探讨人自然杀伤(NK)细胞在CD137抗体作用下通过抗体依赖性细胞毒性作用(ADCC)介导对乳腺癌细胞的杀伤作用。方法NK细胞表型和细胞因子检测实验分组:阴性对照组(未用人CD137抗体处理的NK细胞)、CD137抗体处理组(10μg/mL人CD137抗体处理4 h的NK细胞);NK细胞毒性检测实验分组:根据体系中是否添加NK细胞、人CD137抗体和西妥昔单抗分为8组。流式细胞术检测两组NK细胞表面CD16分子的表达情况,酶联免疫吸附测定(ELISA)法检测两组NK细胞培养上清液中干扰素(IFN)-γ和肿瘤坏死因子(TNF)-α的浓度,乳酸脱氢酶(LDH)法检测8组反应体系中表皮生长因子受体(EGFR)高表达乳腺癌细胞系MDA-MB-231和EGFR低表达乳腺癌细胞系MDA-MB-453的杀伤比例,并采用t检验或析因分析进行统计学分析。结果与阴性对照组比较,CD137抗体处理组CD16+NK细胞比例(79.57﹪±0.92﹪比90.43﹪±0.67﹪)、细胞因子IFN-γ浓度[(388.90±7.02)pg/mL比(523.90±1.90)pg/mL]和TNF-α浓度[(20.59±4.09)pg/mL比(47.22±2.14)pg/mL]均升高,差异有统计学意义(P<0.05);MDA-MB-231细胞杀伤的三因素析因分析结果显示:NK细胞、人CD137抗体和西妥昔单抗3个因素分别对MDA-MB-231细胞的杀伤都有作用(F=5227.276、201.473、1792.242,P均<0.001),3个因素两两之间的交互作用对MDA-MB-231细胞的杀伤也都有作用(F=183.903、1517.187、33.483,P均<0.001),3个因素的二级交互作用差异有统计学意义(F=41.505,P<0.001)。结论人CD137抗体可增强NK细胞分泌细胞毒性因子IFN-γ和TNF-α的能力,同时可上调NK细胞表面CD16分子的表达,从而使得NK细胞可能通过西妥昔单抗介导的ADCC作用增强对表皮生长因子受体(EGFR)高表达乳腺癌细胞的杀伤作用。Objective Investigate the killing effect of NK cells treated by CD137 against breast cancer cells mediated by antibody dependent cell cytotoxicity(ADCC).Methods Groups in phenotype and cytokine detection experiments:negative control group(NK cells not treated with human CD137 antibody),CD137 antibody treatment group(NK cells treated with 10μg/mL human CD137 antibody for 4 h).Groups in cytotoxicity experiment:there are 8 groups according to the addition of NK cells,human CD137 antibody and cetuximab in the system.The ratio of CD16+NK cell was detected by flow cytometry,the concentration of IFN-γand TNF-αin the supernatant of NK cell culture media was detected by enzyme linked immunosorbent assay(ELISA),and the cytotoxicity of NK cells against MDA-MB-231 and MDA-MB-453 cell lines mediated by cetuximab was measured by lactate dehydrogenase(LDH).t test or factorial analysis was applied for statistical analysis.Results Compared with NC group,the ratio of CD16+NK cells and the concentration of IFN-γand TNF-αin the CD137 antibody treatment group was significantly higher than that of the control group(79.57﹪±0.92﹪vs 90.43﹪±0.67﹪,388.90 pg/mL±7.02 pg/mL vs 523.90 pg/mL±1.90 pg/mL,20.59 pg/mL±4.09 pg/mL vs 47.22 pg/mL±2.14 pg/mL,P<0.05).The concentration of IFN-γand TNF-αsecreted from CD137 antibody treated NK cells was(523.90±1.90)pg/mL and(47.22±2.14)pg/mL,higher than that of the control group(388.90±7.02)pg/mL(P<0.01)and(20.59±4.09)pg/mL(P<0.05)respectively.The results of three factor factorial analysis showed that NK cells,human CD137 antibody and cetuximab could kill MDA-MB-231 cells(F=5227.276,201.473,1792.242,P<0.001),and the interaction of the three factors also had effect on the killing of MDA-MB-231 cells(F=183.903,1517.187,33.483,P<0.001).The secondary interaction of the three factors was also statistically significant(F=41.505,P<0.001).Conclusion All above,we have presented that CD137 antibody can enhance the secretion ability of NK cells to secrete IFN-γand TNF-α.Meanwhile,it could u

关 键 词：CD137 抗体 自然杀伤细胞 西妥昔单抗 免疫细胞治疗 表皮生长因子受体 乳腺癌 

分 类 号：R737.9[医药卫生—肿瘤]