乳腺癌源性肿瘤相关成纤维细胞的永生化及鉴定  

Immortalization and identification of cancer associated fibroblasts derived from breast cancer

作　　者：付豪 冯玉梅 FU Hao;FENG Yumei(Department of Biochemistry and Molecular Biology,Tianjin Medical University Cancer Institute and Hospital,Tianjin Medical University,Key Laboratory of Breast Cancer Prevention and Therapy,Ministry of Education,Tianjin′s Clinical Research Center for Cancer,National Clinical Research Center for Cancer,Tianjin 300060,China)

机构地区：[1]天津医科大学肿瘤医院肿瘤研究所生物化学与分子生物学研究室,乳腺癌防治教育部重点实验室,天津市恶性肿瘤临床医学研究中心,国家恶性肿瘤临床医学研究中心,天津300060

出　　处：《天津医科大学学报》2024年第4期289-294,共6页Journal of Tianjin Medical University

基　　金：天津市医学重点学科(专科)建设项目(TJYXZDXK-009A)。

摘　　要：目的:构建可在体外稳定培养和传代的肿瘤相关成纤维细胞(CAFs)并鉴定其生物学特征。方法:取人乳腺浸润性导管癌组织,采用酶解-组织块培养法和差速贴壁法分离纯化CAFs,用免疫荧光检测α-肌动蛋白、血小板源性生长因子受体β和广谱细胞角蛋白;采用人端粒酶表达慢病毒感染CAFs使其永生化;在商品化FGM-2(fibroblast growth medium-2)培养基的基础上添加谷氨酰胺、非必需氨基酸和抗坏血酸以优化培养条件,在体外连续培养传代,并用β-半乳糖苷酶染色鉴定各代细胞中衰老细胞的比例。对构建成功的CAF1、CAF2和CAF3细胞系进行高通量RNA测序,分析差异表达基因(DEGs)及其相关信号通路;基于3个CAF细胞系CAF亚型标志基因的表达水平,鉴定其生物学特征。结果:成功分离原代CAFs,并构建3株永生化CAF细胞系;成功优化了CAFs的培养条件,使CAF细胞系在体外培养时不易发生细胞衰老表型(t=2.972、7.049,均P<0.05);RNA测序结果分析显示,CAF1与CAF2有3150个DEGs,CAF2与CAF3有3544个DEGs;而CAF1与CAF3有343个DEGs。对DEGs进行GO富集分析(gene ontology enrichment analysis)表明,CAF2高表达基因主要参与蛋白水解、细胞外基质重塑、血管生成和细胞黏附等信号通路;CAF1和CAF3高表达基因主要参与细胞分裂、细胞周期、DNA损伤修复和有丝分裂等信号通路。对3个CAF细胞系亚型特征性分析表明,CAF1和CAF 3具有炎性CAFs特征,CAF2具有基质型CAFs特征。结论:成功构建得到3株可在体外稳定传代且具有不同生物学特征的CAF细胞系,为CAFs在乳腺癌发生和进展中作用机制的研究提供了可靠的细胞模型。Objective:To establish cell lines of cancer associated fibroblasts(CAFs)forstable culture and passage in vitro and identify the biological characteristics.Methods:CAFs were isolated from human breast invasive ductal carcinoma tissues using a combination method of enzymatic digestion and explant culture.CAFs were purified using the differential adhesion method.α-Smooth muscle actin,platelet-derived growth factor receptorβand pan-cytokeratin were detected by immunofluorescence.CAFs were immortalized by transinfected human telomerase reverse transcriptase lentivirus.Glutamine,non-essential amino acid,and ascorbic acid were supplemented in commercial fibroblast growth medium-2 medium to optimize culture condition,supporting continuous cultureand passage in vitro.Senescent cell ratio was characterized by usingβX-galactosidase staining.High-throughput RNA sequencing was conducted onsuccessfully constructed CAF1,CAF2 and CAF3 cell lines,and differential expressed genes(DEGs)and related signaling pathways were analyzed.Expression levels of CAF subtype specific genes of the three CAF cell lines were analyzed,and their biological characteristics were identified.Results:Primary CAFs were successfully isolated,and three immortalized CAF cell lines were constructed.Culture conditions for CAFs were successfully optimized to reduce the occurrence of cellular senescence phenotype in vitro.Analysis of RNA sequencing results revealed 3150 DEGs between CAF1 and CAF2,3544 DEGs between CAF2 and CAF3 and 343 DEGs between CAF1 and CAF3.The Gene Ontology enrichment analysis of DEGs showed that highly expressed genes in CAF2 were mainly involved in proteolysis,extracellular matrix remodeling,angiogenesis,and cell adhesion signaling pathways.Highly expressed genes in CAF1 and CAF3 are mainly involved in cell division,cell cycle,DNA damage repair and mitosis signaling pathways.The characteristic analysis of the subtypes of the three CAF cell lines showed that CAF1 and CAF3 exhibited inflammatory CAF characteristics,while CAF2 exhibited

关 键 词：乳腺癌 肿瘤相关成纤维细胞 永生化 RNA测序分析 CAF亚型 

分 类 号：R73-351[医药卫生—肿瘤]