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中华细胞与干细胞杂志(电子版) ›› 2020, Vol. 10 ›› Issue (06) : 346 -353. doi: 10.3877/cma.j.issn.2095-1221.2020.06.005

所属专题： 文献；

论著

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
刘鲁宁1, 陈雪梅1, 马恩奇1, 田清艳1, 刘倩倩1, 刘韬1,†()   
  1. 1. 518001　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
  • 收稿日期:2020-03-06 出版日期:2020-12-01
  • 通信作者: 刘韬
  • 基金资助:
    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、JCYJ201704 12155231633)

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

Luning Liu1, Xuemei Chen1, Enqi Ma1, Qingyan Tian1, Qianqian Liu1, Tao Liu1,†()   

  1. 1. Department of Oncology Rehabilitation, Shenzhen Luohu People's Hospital, the Third Affiliated Hospital of Shenzhen University, Shenzhen 518001, China
  • Received:2020-03-06 Published:2020-12-01
  • Corresponding author: Tao Liu
  • About author:
    Corresponding author: Liu Tao, Email:
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引用本文：

刘鲁宁, 陈雪梅, 马恩奇, 田清艳, 刘倩倩, 刘韬. 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[J/OL]. 中华细胞与干细胞杂志(电子版), 2020, 10(06): 346-353.

Luning Liu, Xuemei Chen, Enqi Ma, Qingyan Tian, Qianqian Liu, Tao Liu. Effects of human CD137 antibody on specific cytotoxicity in NK cells against breast cancer cells in vitro[J/OL]. Chinese Journal of Cell and Stem Cell(Electronic Edition), 2020, 10(06): 346-353.

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（10 μg/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。

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
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 10 μg/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-γ and TNF-α in 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-γ and TNF-α in 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-γ and TNF-α secreted 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-γ and TNF-α. Meanwhile, it could upregulate the expression of CD16 molecules on the surface of NK cells, so that NK cells may enhance the cytotoxicity against epidermal growth factor receptor (EGFR) positive breast cancer cells through ADCC mediated by cetuximab.

Key words: CD137, Antibody, Natural killer cells, Cetuximab, Cellular immunotherapy, EGFR, Breast cancer
表1 乳腺癌细胞杀伤实验分组设计
分组 添加物
NK细胞 人CD137抗体 西妥昔单抗
a - - -
b - + -
c - - +
d - + +
e + - -
f + + -
g + - +
h + + +
图1 倒置显微镜下观察诱导培养的NK细胞（a、c图×100，b、d图×200）
图2 NK细胞诱导培养16 d的生长曲线
图3 培养第16天NK细胞的流式检测
图4 两组NK细胞表面CD16表达比例检测
图5 NK细胞培养上清液中细胞因子浓度比较
图6 倒置荧光显微镜观察乳腺癌细胞MDA-MB-453和MDA-MB-231 （免疫荧光染色，×400）
图7 Western blot检测乳腺癌细胞中EGFR蛋白水平
图8 乳腺癌细胞MDA-MB-453的杀伤比例统计
表2 各组乳腺癌细胞的杀伤比例（ ± s）
分组 实验重复次数 乳腺癌细胞杀伤比例（﹪）
MDA-MB-453 MDA-MB-231
a 3 3.51±1.07 3.95±1.15
b 3 4.59±1.65 4.51±0.53
c 3 5.72±0.06 5.70±0.27
d 3 6.09±0.24 5.69±0.27
e 3 25.08±1.83 15.34±0.08
f 3 31.24±0.06 22.09±1.86
g 3 26.70±0.83 45.34±0.44
h 3 32.20±0.23 62.71±0.79
表3 乳腺癌细胞杀伤实验结果的三因素析因分析结果
因变量：测量值源 MDA-MB-453细胞 MDA-MB-231
F值 P值 F值 P值
NK细胞（A） 2260.154 < 0.001 5227.276 < 0.001
人CD137抗体（B） 42.837 < 0.001 201.473 < 0.001
西妥昔单抗（C） 9.846 0.014 1792.242 < 0.001
A×B 25.891 < 0.001 183.903 < 0.001
A×C 0.319 0.588 1517.187 < 0.001
B×C 0.467 0.514 33.483 < 0.001
A×B×C 0.001 0.981 41.505 < 0.001
图9 乳腺癌细胞MDA-MB-231的杀伤比例统计图
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