乳腺癌间质干细胞通过IL-6-STAT3信号通路影响乳腺癌MCF-7细胞增殖和迁移  

Effects of breast cancer mesenchymal stem cells on proliferation and migration of breast cancer MCF-7 cells via IL-6-STAT3 signaling pathway

作　　者：周颖[1] 徐会涛[1] 张欢欢[1] 张楚 郑萍[1] 杨晋[1] Zhou Ying;Xu Huitao;Zhang Huanhuan;Zhang Chu;Zheng Ping;Yang Jin(Department of Laboratory Medicine,the First People's Hospital of Lianyungang,Lianyungang 222002,China)

机构地区：[1]连云港市第一人民医院检验科,连云港222002

出　　处：《肿瘤研究与临床》2023年第11期801-807,共7页Cancer Research and Clinic

基　　金：连云港市卫生健康委员会医学创新团队建设项目(2018)。

摘　　要：目的探讨乳腺癌间质干细胞(BC-MSC)对乳腺癌MCF-7细胞增殖与迁移能力的影响及相关机制。方法取乳腺癌患者术后切除癌组织和癌旁组织及健康人骨髓标本,采用组织贴壁法,加入成骨、成脂诱导液诱导分化,分离培养BC-MSC、乳腺癌旁间质干细胞(BCN-MSC)和健康人骨髓间质干细胞(BM-MSC),流式细胞术检测其表面标志。收集BC-MSC、BCN-MSC与健康人BM-MSC培养48 h的上清液,分别用于培养MCF-7细胞,分别为BC-MSC组、BCN-MSC组、BM-MSC组,对照组为常规培养的MCF-7细胞。采用四甲基偶氮唑盐(MTT)法检测各组MCF-7细胞的增殖能力,平板克隆实验检测MCF-7细胞的克隆形成能力,Transwell实验检测MCF-7细胞的迁移能力,实时荧光定量聚合酶链反应(qRT-PCR)检测MCF-7细胞白细胞介素(IL)6及上皮间质转化(EMT)相关基因(E-cadherin、vimentin、snail)mRNA的相对表达水平,蛋白质印迹法检测MCF-7细胞p-STAT3、E-cadherin、vimentin、snail蛋白的表达,Luminex液态芯片技术检测不同间质干细胞(MSC)培养上清中细胞因子水平。将IL-6中和抗体加入BC-MSC培养上清中,用上清培养MCF-7细胞(BC-MSC+IL-6中和抗体组),检测MCF-7细胞增殖与迁移能力及相关基因、蛋白表达的变化。结果成功分离BC-MSC、BCN-MSC、BM-MSC;BC-MSC表面CD29、CD44、CD90阳性表达,CD14、CD34、CD45阴性表达,符合MSC特征。MTT法检测结果显示,对照组、BC-MSC组、BCN-MSC组和BM-MSC组培养48 h的MCF-7细胞的吸光度值分别为0.31±0.02、0.54±0.03、0.43±0.02和0.42±0.02,差异有统计学意义(F=56.52,P<0.05);平板克隆实验结果显示,4组每个平皿克隆数分别为(180±9)个、(439±17)个、(319±16)个和(306±19)个,差异有统计学意义(F=222.70,P<0.05);Transwell实验结果显示,4组穿膜细胞数分别为(6.5±1.0)个、(23.2±2.4)个、(16.0±1.3)个和(14.8±2.0)个,差异有统计学意义(F=49.44,P<0.05);qRT-PCR检测显示,4组IL-6 mRNA相对表达量分别为1.07±0.11、13.7Objective To explore the effects of breast cancer mesenchymal stem cells(BC-MSC)on the proliferation and migration of breast cancer MCF-7 cells and the related mechanisms.Methods The resected cancer tissues and paracancerous tissues were taken from breast cancer patients after surgery,and the bone marrow samples of healthy people were selected.BC-MSC,breast cancer paracancerous mesenchymal stem cells(BCN-MSC)and bone marrow mesenchymal stem cells(BM-MSC)of healthy people were isolated and cultured by tissue adhesion method,and their differentiation ability was induced by the addition of osteogenic and lipogenic induction,and their surface markers were detected by flow cytometry.The supernatants of BC-MSC,BCN-MSC and BM-MSC of healthy people cultured for 48 h were collected and used for the culture of MCF-7 cells as BC-MSC group,BCN-MSC group and BM-MSC group,respectively,and the control group was the conventional cultured MCF-7 cells.The proliferation ability of MCF-7 cells in each group was detected by methyl thiazol tetrazolium(MTT)assay,the clone formation ability of MCF-7 cells was detected by plate cloning assay,the migration ability of MCF-7 cells was detected by Transwell assay,and the mRNA relative expressions of interleukin(IL)-6 and epithelial mesenchymal transition(EMT)-related genes(E-cadherin,vimentin,snail)were detected by quantitative real-time fluorescence polymerase chain reaction(qRT-PCR)in MCF-7 cells.Western blotting was used to detect expressions of p-STAT3,E-cadherin,vimentin and snail proteins in MCF-7 cells.Luminex liquid microarray technology was used to detect cytokine levels in culture supernatants of different mesenchymal stem cells(MSC).IL-6 neutralizing antibody was added into the supernatant of BC-MSC,MCF-7 cells were cultured with the supernatant(BC-MSC+IL-6 neutralizing antibody group),and then the proliferation and migration abilities of MCF-7 cells were tested,as well as the expression changes of related genes and proteins.Results BC-MSC,BCN-MSC and BM-MSC were successfully isol

关 键 词：乳腺肿瘤 间质干细胞 STAT3转录因子 白细胞介素6 细胞增殖 细胞迁移 上皮-间质转化 

分 类 号：R737.9[医药卫生—肿瘤]