人乳腺癌原代细胞系的建立及其对药物的敏感性  

Establishment of primary cell lines from human breast cancer and analysis of their sensitivity to drugs

作　　者：罗钰蓓 黄建军 杨文秀 张军红 郝弦 万宇 李青 李静[5] 赵春华[5] 豆晓伟 LUO Yubei;HUANG Jianjun;YANG Wenxiu;ZHANG Junhong;HAO Xian;WAN Yu;LI Qing;LI Jing;ZHAO Chunhua;DOU Xiaowei(Department of Pathology,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Clinical Research Center,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Breast Surgery,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Department of Emergency Orthopedics,the Affiliated Hospital of Guizhou Medical University,Guiyang 550004,Guizhou,China;Institute of Basic Medical Sciences Chinese Academy of Medical Sciences,School of Basic Medicine,Peking Union Medical College&Center of Excellence in Tissue Engineering,Chinese Academy of Medical Sciences&Beijing Key Laboratory of New Drug Development and Clinical Trial of Stem Cell Therapy,Beijing 100005,China)

机构地区：[1]贵州医科大学附属医院病理科,贵州贵阳550004 [2]贵州医科大学附属医院临床医学研究中心,贵州贵阳550004 [3]贵州医科大学附属医院乳腺外科,贵州贵阳550004 [4]贵州医科大学附属医院急诊骨科,贵州贵阳550004 [5]中国医学科学院基础医学研究所北京协和医学院基础学院&中国医学科学院组织工程研究中心&干细胞新药研发及临床转化研究北京市重点实验室,北京100005

出　　处：《贵州医科大学学报》2024年第12期1780-1786,1793,共8页Journal of Guizhou Medical University

基　　金：贵州省科技计划项目重点项目(ZK[2023]重点033)。

摘　　要：目的探讨人乳腺癌(BC)原代细胞系的培养建立并分析其药物敏感性。方法取BC浸润性导管癌患者的BC组织4份,Ⅱ型胶原酶消化并培养于BC第1代培养基(BCMⅠ),细胞快速增殖时更换为杜氏改良Eagle培养基(DMEM),利用倒置显微镜观察细胞形态,明确是否成功培养无限传代的4种BC原代细胞系;采用短串联重复序列(STR)分析上述4种原代细胞系的特异性遗传标记;取对数生长期的4种BC原代细胞系,分为50 nmol/L多柔比星(Doxorubicin)组、50 nmol/L吡柔比星(Pirarubicin)组及对照(Control)组(不加药物),采用CellTiter法检测细胞活力判断对其药物的敏感性;收集上述细胞来源的4例BC浸润性导管癌患者的一般临床资料、病理资料[组织学类型、年龄、肿瘤大小、肿瘤淋巴结转移(TNM)分期、病理分级、淋巴结转移及月经情况等]及新辅助、术后辅助治疗方案信息,采用苏木精-伊红染色(HE)检测BC组织的形态学特征及BC分型,采用免疫组织化学(IHC)和荧光原位杂交(FISH)观测BC组织中雌激素受体(ER)、孕激素受体(PR)、人表皮生长因子受体2(HER-2)、雄激素受体(AR)、细胞角蛋白5/6(CK5/6)、增值细胞核抗原(Ki-67)、拓扑异构酶Ⅱ(TOP-Ⅱ)分子及HER-2基因的表达。结果成功培养了4种永生化的乳腺癌原代细胞系,分别为BC8#、BC11#、BC19#及BC21#;STR检测4株乳腺癌原代细胞为没有交叉污染的人来源细胞系,这些乳腺癌原代细胞系有明确的临床病理特点、病理标志物检测、临床诊断及治疗方案结果及CellTiter-Glo试剂盒检测结果显示,50 nmol/L Doxorubicin可以不同程度的抑制BC8#、BC11#、BC19#及BC21#细胞生长(P<0.05);50 nmol/L Pirarubicin不同程度地抑制BC11#和BC19#细胞生长,但对BC8#和BC21#细胞生长没有作用(P<0.05)。结论成功培养了4种永生化的乳腺癌原代细胞系,这4种乳腺癌原代细胞系对化疗药物Doxorubicin和Pirarubicin的反应不同。Objective To establish primary cell lines from human breast cancer(BC)and analyzing their drug sensitivity.Methods BC tissues were obtained from four patients diagnosed as invasive ductal carcinoma.The tissues were digested with typeⅡcollagenase and cultured in BC first generation culture medium(BCMⅠ).When the cells proliferated rapidly,the medium was switched to Dulbecco's modified Eagle medium(DMEM).The cell morphology was observed using an inverted microscope to clarify whether 4 BC primary cell lines with unlimited passages had been successfully cultured.Short tandem repeat(STR)analysis was used to identify specific genetic markers in the 4 primary cell lines mentioned above.Four primary BC cell lines at logarithmic growth phase were selected and divided into doxorubicin group(50 nmol/L),pirarubicin group(50 nmol/L)and control group(without medication).CellTiter assay was used to examine cell viability and assess drug sensitivity.Four aforementioned cell sources of 4 patients with invasive ductal carcinoma were collected,including general clinical and pathological data[histological type,age,tumor size,tumor lymph node metastasis(TNM)stage,pathological grading,lymph node metastasis and menstrual status,etc.]as well as the information on neoadjuvant and postoperative adjuvant treatment plans.Hematoxylin&eosin(H&E)staining was used to detect the morphological characteristics and BC types of BC tissue.Immunohistochemistry(IHC)and fluorescence in situ hybridization(FISH)were used to observe the expressions of estrogen receptor(ER),progesterone receptor(PR),human epidermal growth factor receptor 2(HER-2),androgen receptor(AR),cytokeratin 5/6(CK5/6),Ki-67,topoisomeraseⅡ(TOP-Ⅱ)molecules,and HER-2 genes in BC tissues.Results Four immortalized primary breast cancer cell lines were successfully cultivated,namely BC8#,BC11#,BC19#,and BC21#.STR verified that the four primary breast cancer cell lines were human origin cell lines without cross contamination.These primary breast cancer cell lines had clear clinicopat

关 键 词：乳腺癌 原代细胞系 培养体系 药物预测 新癌症模型 CellTiter-Glo荧光细胞活力实验 

分 类 号：R737.9[医药卫生—肿瘤]