双三体胚胎干细胞的增殖与分化特征分析  

Proliferation and Differentiation Characteristics of Double-Trisomic Embryonic Stem Cells

作　　者：张美丽[1] 肖蓉 贾玉艳 黄粤[1] ZHANG Meili;XIAO Rong;JIA Yuyan;HUANG Yue(State Key Laboratory of Medical Molecular Biology,Institute of Basic Medical Sciences Chinese Academy of Medical Sciences,School of Basic Medicine Peking Union Medical College,Beijing 100005,China)

机构地区：[1]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,医学分子生物学国家重点实验室,北京100005

出　　处：《中国细胞生物学学报》2021年第5期922-931,共10页Chinese Journal of Cell Biology

基　　金：中国医学科学院医学与健康科技创新工程项目(批准号:2016-I2M-3-002)资助的课题。

摘　　要：该文旨在研究双三体胚胎干细胞(embryonic stem cells,ESCs)在细胞增殖、分化以及畸胎瘤形成等方面的特征,揭示非整倍体与肿瘤发生之间的关系。首先建立了两株常染色体双三体的小鼠ESC株系,通过微阵列比较基因组杂交(array comparative genomic hybridization,array CGH)和荧光原位杂交(fluorescence in situ hybridization,FISH)实验对双三体ESC株系进行了染色体拷贝数分析和核型鉴定;通过绘制细胞生长曲线检测了双三体ESCs的增殖能力;通过流式细胞术检测了双三体ESCs的细胞周期和细胞凋亡情况;采用细胞克隆形成实验分析了双三体ESCs的克隆形成效率;通过实时荧光定量PCR和免疫荧光染色实验检测了双三体ESCs中多能干细胞标志物的表达;通过撤掉培养体系中的白血病抑制因子(leukemia inhibitory factor,LIF)诱导分化和进行拟胚体(embryoid body,EB)形成实验检测了双三体ESCs的分化能力;通过重度联合免疫缺陷(severe combined immunodeficiency,SCID)小鼠皮下接种细胞实验分析了双三体ESCs的畸胎瘤形成能力和体内分化能力。结果显示,这两株双三体细胞分别是3号与6号染色体双三体并伴有Y染色体丢失的ESCs(DTs-3+6),以及6号与8号染色体双三体的ESCs(DTs-6+8)。双三体ESCs表现出相对于野生型细胞较强的生长增殖能力和OCT4、SOX2、NANOG等多能干细胞标志物的高表达。当培养液中不添加LIF时,野生型细胞基本完全走向分化,而双三体细胞形成许多未分化或部分分化的克隆,碱性磷酸酶(alkaline phosphatase,AP)染色阳性。在EB分化早期,双三体细胞中Fgf5、T、Foxa2等三胚层标志物的表达水平较野生型细胞明显降低,分化滞后。当被接种到SCID小鼠皮下后,双三体ESCs形成畸胎瘤的能力较野生型细胞增强,畸胎瘤中包含大量未分化区域。因此,双三体ESCs的生长增殖能力增强,它通过限制细胞分化能力而促进畸胎瘤形成。双三体ESCs�This study aims to investigate the proliferation,differentiation,and teratoma formation characteristics of double-trisomic ESCs(embryonic stem cells),and discover the relationships between aneuploidy and tumorigenesis.Two lines of autosomal double-trisomic mouse ESCs were established.Array CGH(array comparative genomic hybridization)and FISH(fluorescence in situ hybridization)were used to determine the chromosome copy number variations and the karyotyping of the two double-trisomic ESC lines.Cell growth curves were made to evaluate the proliferation abilities of the double-trisomic ESCs.Flow cytometry was used to detect the cell cycle distributions and the levels of apoptosis in double-trisomic ESCs.Colony-forming assays were performed to evaluate the colony formation efficiencies of double-trisomic ESCs.qRT-PCR and immunofluorescence analyses were conducted to determine whether the pluripotency markers were normally expressed in double-trisomic ESCs.Moreover,LIF(leukemia inhibitory factor)withdrawal and EB(embryoid body)formation assays were performed to evaluate the in vitro differentiation status of these double-trisomic ESCs.Teratoma assays were conducted by using SCID(severe combined immunodeficiency)mice to determine the effects of double-trisomies on teratoma formation and the differentiation capacities in vivo.Array CGH and FISH experiments showed that one cell line gained extra chromosome 3 and chromosome 6 but lost chromosome Y,which was named as DTs-3+6.Another cell line had extra chromosomes of 6 and 8,which was named as DTs-6+8.Double-trisomic ESCs exhibited rapid proliferation characteristics when compared with wild-type ESCs.They expressed stem cell markers OCT4,SOX2 and NANOG when cultured under normal ESC culture conditions.Upon LIF withdrawal,wild-type ESCs mostly went to total differentiation,while double-trisomic ESCs formed many partially differentiated or undifferentiated clones,which were positive for AP(alkaline phosphatase)staining.In the early stage of EB differentiation,the expression o

关 键 词：非整倍体 双三体 胚胎干细胞 增殖 分化 畸胎瘤 

分 类 号：R730.2[医药卫生—肿瘤]