基于CD44/CD24和ALDH1对人乳腺癌干细胞分离鉴定的方法学研究  

Methodological study on the isolation and identification of human breast cancer stem cells based on CD44 and ALDH1

作　　者：蒋琴 张曼俐 陈洪清 张旭帆 李盛 JIANG Qin;ZHANG Manli;CHEN Hongqing;ZHANG Xufan;LI Sheng(Laboratory Department of Mianyang Traditional Chinese Medicine Hospital(Mianyang Hospital of Chengdu University of Traditional Chinese Medicine),Sichuan Mianyang 621000,China;Department of Laboratory Medicine,Hospital of Chengdu University of Traditional Chinese Medicine,Sichuan Chengdu 610075,China.)

机构地区：[1]绵阳市中医医院(成都中医药大学附属绵阳医院)检验科,四川绵阳621000 [2]成都中医药大学附属医院检验科,四川成都610075

出　　处：《现代肿瘤医学》2024年第15期2722-2729,共8页Journal of Modern Oncology

摘　　要：目的:通过分子、细胞实验、动物实验三方面研究分别从四类乳腺癌组织样本寻找分离和鉴定筛选乳腺癌干细胞的方法,旨在为乳腺癌干细胞的研究寻找确定的方法,为乳腺癌的治疗提供参考方向。方法:采用DocSense组织单细胞悬液制备方法从乳腺癌组织中获取原代乳腺癌单细胞用于细胞培养,通过采用CD44/CD24和乙醛脱氢酶1(ALDH1)抗体进行流式分选出CD44^(+)/CD24^(-/low)组、ALDH1+组及非干细胞表型CD44-/ALDH1-组的细胞群,分组后的细胞采用乳腺癌干细胞干性鉴定(微球克隆形成实验),MTT实验分别绘制Luminal A型,Luminal B型,HER-2+型,Basal-like型筛选出的CD44^(+)/CD24^(-/low)组、ALDH1+组和CD44-/ALDH1-组细胞生长曲线,最后采用裸鼠成瘤实验及剥离肿瘤组织病理切片采用苏木精-伊红法染色(HE染色)验证所分选出来细胞干性及组织为乳腺癌组织。结果:本研究利用组织单细胞消融技术、干细胞微球形成实验及流式细胞术,成功富集并筛选出4类分子亚型的8种乳腺癌肿瘤干细胞;通过肿瘤微球克隆形成实验、MTT实验鉴定出标记CD44^(+)/CD24^(-/low)、ALDH1+表型的乳腺癌干细胞侵袭性和增殖能力更强;裸鼠皮下荷瘤HE染色进一步证明CD44^(+)/CD24^(-/low)、ALDH1+表型的乳腺癌细胞成瘤能力更强、异质性更明显。结论:CD44^(+)/CD24^(-/low)组、ALDH1+组乳腺癌干细胞干性表达能力较强,可作为乳腺癌干细胞的鉴定和筛选的关键指标,为乳腺癌的治疗提供新的参考方向。Objective:To isolate and identify breast cancer stem cells from breast cancer tissues by molecular experiments,cell experiments,and animal experiments from four types of breast adenocarcinoma tissue samples.Aiming to find a treatment method for the study of breast cancer stem cells and provide directions for the treatment of breast cancer.Methods:Primary breast cancer single-cell suspension was obtained by DocSense tissue single-cell suspension preparation system for cell culture.CD44^(+)/CD24^(-/low) group,ALDH1+group and non stem cell phenotype CD44-/ALDH1-group were selected by flow cytometry using CD44/CD24 and ALDH1 antibodies.The MTT experiment draws the growth curves of the CD44^(+)/CD24^(-/low),ALDH1+group,and CD44-/ALDH1-group screened out for Luminal A type,Luminal B type,HER-2+type,and Basal-like type respectively.Finally,nude mouse tumorigenesis experiments were performed,pathological sections of tumor tissue were peeled off,and hematoxylin-eosin staining(HE staining)was used to verify the stemness of the sorted cells and that the tissue was breast cancer tissue.Results:This study employed tissue single-cell ablation technology,stem cell tumor microsphere formation experiments,and flow cytometry,successfully enriching and identifying eight breast cancer stem cells across four molecular subtypes.Through microsphere clonal formation assays and MTT assays,breast cancer stem cells marked with the CD44^(+)/CD24^(-/low),ALDH1+phenotype were found to have stronger invasiveness and proliferative abilities.Subcutaneous tumor implantation in nude mice followed by HE staining further demonstrated that breast cancer cells with the CD44^(+)/CD24^(-/low),ALDH1+phenotype exhibit enhanced tumorigenic capacity and pronounced heterogeneity.Conclusion:The CD44^(+)/CD24^(-/low) and ALDH1+groups have stronger stem cell expression in breast cancer,which can be used as the research direction for identifying and treating breast cancer stem cells.

关 键 词：乳腺癌干细胞 流式分选 培养 成瘤分析 鉴定 

分 类 号：R737.9[医药卫生—肿瘤]