新型VEGF单克隆抗体与全反式维甲酸联合应用对乳腺癌MCF-7细胞增殖的抑制作用

doi:10.13481/j.1671-587X.20210213

摘要/Abstract

摘要： 目的

探讨新型血管内皮生长因子（VEGF）单克隆抗体、全反式维甲酸（ATRA）、新型VEGF单克隆抗体与ATRA联合用药对乳腺癌MCF-7细胞的影响，为乳腺癌的临床治疗提供新方案。

方法

将MCF-7乳腺癌细胞分为对照组（不做任何处理）、VEGF抗体组（20 mg·L^－1VEGF抗体）、ATRA组（10 μmol·L^－1ATRA）和VEGF抗体+ATRA组（20 mg·L^－1VEGF抗体+10 μmol·L^－1 ATRA）4组，每组5个重复。加入不同药物处理MCF-7细胞48 h后，采用ELISA法检测新型VEGF抗体浓度，MTT法检测MCF-7细胞增殖率，流式细胞术检测MCF-7细胞凋亡率，免疫印迹法检测MCF-7细胞中B细胞淋巴瘤2（Bcl-2）、Bcl-2相关X蛋白（Bax）和核转录因子κB（NF-κB）蛋白表达水平。

结果

ELISA法检测，新型人类重组VEGF单克隆抗体浓度为200 μg·L^－1，与阳性抗体对照比较其质量良好。MTT法检测，与对照组比较，VEGF抗体+ATRA组MCF-7细胞增殖率明显降低（P<0.01）。流式细胞术检测，与对照组、VEGF抗体组和ATRA组比较，VEGF抗体+ATRA组MCF-7细胞凋亡率明显升高（P<0.01）；与对照组比较，VEGF抗体组和ATRA组MCF-7细胞凋亡率明显升高（P<0.01）；与VEGF抗体组比较，ATRA组MCF-7细胞凋亡率升高（P<0.01）。免疫印迹法检测，与对照组、VEGF抗体组和ATRA组比较，VEGF抗体+ATRA组MCF-7细胞中Bcl-2蛋白表达水平降低（P<0.05）；与对照组和VEGF抗体组比较，ATRA组MCF细胞中NF-κB蛋白表达水平明显降低（P<0.05或P<0.01）， Bax蛋白表达水平明显升高（P<0.01）。

结论

新型VEGF单克隆抗体和ATRA可以抑制乳腺癌MCF-7细胞增殖、促进细胞凋亡，而二者联合用药的效果要显著优于单独用药。

关键词: 乳腺肿瘤, 血管内皮生长因子, 单克隆抗体, 全反式维甲酸, 细胞凋亡, 细胞增殖

Abstract: Objective

To explore the effects of new vascular endothelial growth factor （VEGF） monoclonal antibody， all-trans retinoic acid （ATRA）， new VEGF monoclonal antibody combined with ATRA on the breast cancer MCF-7 cells， and to provide a new clinical approach for the treatment of breast cancer.

Methods

The breast cancer MCF-7 cells were divided into control group （without any treatment）， VEGF antibody group （20 mg·L^－1VEGF antibody）， ATRA group （10 μmol·L^－1 ATRA） and VEGF antibody + ATRA group （20 mg·L^－1 VEGF antibody+10 μmol·L^－1 ATRA）， each with 5 replicates. After the MCF-7 cells were treated with different drugs for 48 h，the VEGF antibody concentration was determined by ELISA， the proliferation rate of MCF-7 cells was determined by MTT assay， the apoptotic rate of MCF-7 cells was determined by flow cytometry， and the expression levels of B cell lymphoma-2（Bcl-2），Bcl-2 associated X protein（ Bax） and nuclear factor-κB（NF-κB） proteins in the MCF-7 cells were determined by Western blotting method.

Results

The ELISA results showed that the new human recombinant VEGF monoclonal antibody had good quality compared with positive antibody control and its concentration was 200 μg·L^-1.The MTT results showed that the prolifenation rate of MCF-7 cells in VEGF antibody + ATRA group was decreased obviously compared with control group （P<0.01）.The flow cytometry results showed that compared with control group，VEGF antibody group and ATRA group， the apoptotic rate of MCF-7 cells in VEGF antibody + ATRA group was significantly increased （P<0.01）；compared with control group，the apoptotic rates of MCF-7 cells in VEGF antibody group and ATRA group were significantly increased（P<0.01）；compared with VEGF antibody group，the apototic rate of MCF-7 cells in ATRA group was significantly increased（P<0.01）. The results of Western blotting method showed that compared with control group， VEGF antibody group and ATRA group， the expression level of Bcl-2 protein in VEGF antibody+ATRA group was significantly decreased （P<0.05）；compared with control group and VEGF antibody group， the expression level of NF-κB protein in MCF-7 cells in ATRA group was decreased （P<0.05 or P<0.01）， and the expression level of Bax protein was increased （P<0.01）.

Conclusion

The new VEGF monoclonal antibody and ATRA can inhibit the cell proliferation and promote apoptosis of breast cancer MCF-7 cells， and the effect of their combination is significantly better than each drug used alone.

Key words: breast neoplasms, vascular endothelial growth factor, monoclonal antibody, all-trans retinoic acid, apoptosis, proliferation

中图分类号:

R737.9

唐浩轩,孟楠峰,杜美玲,王伟,何涛. 新型VEGF单克隆抗体与全反式维甲酸联合应用对乳腺癌MCF-7细胞增殖的抑制作用[J]. 吉林大学学报(医学版), 2021, 47(2): 344-351.

Haoxuan TANG,Nanfeng MENG,Meiling DU,Wei WANG,Tao HE. Inhibitory effect of new VEGF monoclonal antibody combined with all-trans retinoic acid on proliferation of breast cancer MCF-7 cells[J]. Journal of Jilin University(Medicine Edition), 2021, 47(2): 344-351.

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Group	A value	Protein concentrasion[ρ_B/（μg·L^－1）]
VEGF-antibody	1.879±0.045	200
Positive antibody standard	1.119±0.060	125

Group	Cytotoxicity
Control	0.30±0.013
1 μmol·L^－1 ATRA+15 mg·L^－1 VEGF-antibody	0.32±0.011
5 μmol·L^－1 ATRA+15 mg·L^－1 VEGF-antibody	0.32±0.005
1 μmol·L^－1 ATRA+20 mg·L^－1 VEGF-antibody	0.34±0.011^*
5 μmol·L^－1 ATRA+20 mg·L^－1 VEGF-antibody	0.36±0.014^*
10 μmol·L^－1 ATRA+10 mg·L^－1 VEGF-antibody	0.36±0.020^*
10 μmol·L^－1 ATRA+15 mg·L^－1 VEGF-antibody	0.38±0.020^**
15 μmol·L^－1 ATRA+10 mg·L^－1 VEGF-antibody	0.43±0.018^**
10 μmol·L^－1 ATRA+20 mg·L^－1 VEGF-antibody	0.51±0.019^**

Group	Proliferation rate
Control	48.02±2.95
VEGF-antibody	37.26±1.11^*
ATRA	34.22±0.75^*
ATRA+VEGF-antibody	31.12±0.36^*△#

Group	Apoptotic rate
Control	6.25±1.28
VEGF-antibody	14.60±0.39^*
ATRA	19.75±1.82^*
ATRA+VEGF-antibody	31.35±1.73^*△#

Group	Bcl-2 protein	Bax protein	NF-κB protein
Control	0.139 4±0.007 6	0.191 9±0.016 4	0.174 9±0.017 9
VEGF-antibody	0.122 2±0.007 6^**	0.379 4±0.052 6^**	0.172 9±0.014 5
ATRA	0.096 5±0.005 8^**	0.646 0±0.065 5^**△△	0.117 6±0.004 9^*△△
ATRA+VEGF-antibody	0.079 0±0.006 0^**△△#	0.844 2±0.072 7^**△△	0.129 8±0.005 9^*△
P	<0.01	<0.01	<0.05