基于蛋白质组学技术探讨麻疹减毒活疫苗191株抑制三阴性乳腺癌细胞增殖的可能机制  

Possible mechanism of 191 measles attenuated live vaccine strains inhibiting the proliferation of triple negative breast cancer MDA-MB-231 cells based on proteomics technology

作　　者：姚梦玮 李玉迁 徐庆胜 方小菡 魏洪 迟茜文[3] 刘蕊 YAO Mengwei;LI Yuqian;XU Qingsheng;FANG Xiaohan;WEI Hong;CHI Qianwen;LIU Rui(a.Department of Microbiology,Basic Medical College,Guizhou Medical University,Guiyang 550025,Guizhou,China;Key Laboratory of Microbiology and Parasitology of Education Department of Guizhou Province,Guizhou Medical University,Guiyang 550025,Guizhou,China;National Experimental Teaching Center for Basic Medicine,Guizhou Medical University,Guiyang 550025,Guizhou,China;Experimental Center of Hongyan District,the Second Affiliated Hospital of Guizhou University of Traditional Chinese Medicine,Guiyang 550003,Guizhou,China)

机构地区：[1]贵州医科大学基础医学院微生物学教研室,贵州贵阳550025 [2]贵州医科大学贵州省普通高等学校病原生物学特色重点实验室,贵州贵阳550025 [3]贵州医科大学基础医学国家级实验教学中心,贵州贵阳550025 [4]贵州中医药大学第二附属医院红岩院区中心实验室,贵州贵阳550003

出　　处：《中国肿瘤生物治疗杂志》2023年第7期568-576,共9页Chinese Journal of Cancer Biotherapy

基　　金：贵州省卫生健康委科学技术基金(No.GZWKJ2023-280);2021年国家级大学生创新创业训练项目(No.202110660028);2020年省级大学生创新训练项目(No.S202010660053);贵州省教育厅平台项目(No.黔教技[2022]019号)。

摘　　要：目的:基于蛋白质组学技术探讨麻疹减毒活疫苗191株(MV-Hu191)在体内外对三阴性乳腺癌MDA-MB-231、4T1细胞的影响及其作用机制。方法:采用CCK-8法检测MV-Hu191对MDA-MB-231和4T1细胞增殖的影响;液相色谱-质谱联用技术分析MV-Hu191处理对MDA-MB-231细胞中蛋白质谱的影响,多重数据库筛选蛋白质谱中的典型差异蛋白质并进行GO、KEGG、亚细胞定位与功能注释。瘤内注射1×106 TCID50 MV-Hu191干预4T1细胞移植瘤模型小鼠,流式细胞术检测小鼠脾组织中T细胞亚群,ELISA法检测小鼠血清TNF-α和IL-6含量。结果:体外实验结果表明,MV-Hu191具有抑制MDA-MB-231和4T1细胞增殖的作用,差异均具有统计学意义(P<0.01)。蛋白质组学分析结果显示,MV-Hu191作用MDA-MB-231细胞后明显上调蛋白质有38个、下调有12个;差异表达的蛋白质主要参与细胞黏附、信号受体激活、细胞代谢、应激反应等生物学过程,22个差异蛋白质亚细胞定位位于细胞外,KEGG功能分类显示与免疫调节功能相关的差异蛋白质最多且均为上调蛋白,包括C4A、C8B、SERPINF2、A2M、SERPINC1、CTSB、SERPING1、C5;PPI预测发现免疫相关差异蛋白与CD4、CD8、TNF-α及IL-6相互关联。体内实验结果显示,MV-Hu191干预组小鼠脾组织中CD4+T细胞数量略高于对照组,但差异无统计学意义(P>0.05),CD4+/CD8+T细胞比值明显高于对照组(P<0.05),血清TNF-α和IL-6含量显著上升(均P<0.01)。结论:MV-Hu191显著抑制MDA-MB-231、4T1细胞增殖及拮抗4T1细胞荷瘤小鼠成瘤性,其机制可能是MV-Hu191通过激活免疫效应分子实现抗肿瘤作用。Objective:To investigate the effect and mechanism of live attenuated measles vaccine strain 191(MV-Hu191)on triple negative breast cancer MDA-MB-231 and 4T1 cells in vitro and in vivo based on proteomics.Methods:CCK-8 method was used to analyze the effect of MV-Hu191 on the proliferation of MDA-MB-231 and 4T1 cells.Liquid chromatography-mass spectrometry was used to analyze the effect of MV-Hu191 treatment on protein spectrum in MDA-MB-231 cells.Multiple databases were used to screen typical differentially expressed proteins,followed with GO,KEGG,subcellular localization and functional annotation.Intratumoral injection of 1×10^(6) TCID50 MV-Hu191 was used to intervene the growth of 4T1 cell transplanted tumor in mice.Flow cytometry was applied to detect T cell subpopulations in splenic tissue,and ELISA was used for the detection of serum levels of TNF-αand IL-6.Results:In vitro experiments showed that MV-Hu191 could significantly inhibit the proliferation of MDA-MB-231 and 4T1 cells(P<0.01).Proteomic analysis showed that 38 proteins were significantly upregulated while 12 proteins were downregulated in MDA-MB-231 cells after MV-Hu191 treatment.The differentially expressed proteins were mainly involved in the biological processes of cell adhesion,signaling receptor activation,cell metabolism,and stress responses.The subcellular localization of 22 differentially expressed proteins was located outside the cell.The KEGG functional classification showed that the most differentially expressed proteins were related to immunomodulatory functions and they were all upregulated,including C4A,C8B,SERPINF2,A2M,SERPINC1,CTSB,SERPING1,and C5;PPI prediction found immune-related differential proteins were associated with CD4,CD8,TNF-ɑand IL-6.In vivo experiments showed that the number of CD4^(+)T cells in spleen tissues of mice in the MV-Hu191 intervention group was higher than that in the control group,but the difference was not significant(P>0.05).The ratio of CD4+/CD8+T cells and the serum levels of TNF-ɑand IL-6 in the MV

关 键 词：麻疹溶瘤病毒 MV-Hu191 三阴性乳腺癌 MDA-MB-231细胞 4T1细胞 细胞增殖 蛋白质组学 

分 类 号：R737.9[医药卫生—肿瘤] R730.5[医药卫生—临床医学]