摘要
目的富集裸鼠乳腺癌移植瘤中的乳腺癌干细胞并进行分选。方法将人乳腺癌MDA-MB-231细胞接种于20只裸鼠右侧腋窝皮下,观察肿瘤的生长情况,30 d后剥离肿瘤,对组织进行HE染色,并从组织中分离出肿瘤细胞。用含血清的DMEM培养液培养分离出的移植瘤细胞,观察移植瘤细胞的形态及生长情况;应用流式细胞分选仪检测移植瘤细胞中干细胞(CD44^(+)CD24^(-/low)细胞)比例。用含表皮生长因子、碱性成纤维细胞生长因子和B27细胞培养添加剂的无血清DMEM培养液培养移植瘤细胞,获得细胞微球体;应用流式细胞分选仪检测培养第10天微球体中干细胞比例,并根据细胞表面标志分选出干细胞。结果20只裸鼠皮下注入MDA-MB-231细胞9 d后,17只皮下出现肿瘤,肿瘤组织实质细胞多,间质少,瘤细胞呈条索样排列,细胞体积大,胞质丰富,细胞核大小不一,深染,核分裂象多见,核仁清晰,多形性明显。用含血清的DEME培养液培养移植瘤细胞24 h后,细胞开始贴壁,3 d后贴壁率达60%,7 d后细胞增殖加快,细胞形态更趋于一致,多呈梭形,和MDA-MB-231细胞形态无明显差异,其中干细胞比例为(0.10±0.02)%。用含细胞培养添加剂的无血清培养液培养移植瘤细胞后,细胞呈球样生长,第10天微球体中干细胞比例为(70.47±2.03)%。结论裸鼠皮下接种MDA-MB-231细胞能建立裸鼠乳腺癌异位移植瘤模型,分离出的移植瘤细胞经无血清培养能富集移植瘤中的乳腺癌干细胞,从而能分选出更多乳腺癌干细胞,为研究乳腺癌干细胞生物学特性提供基础。
Objective Enriching and isolating breast cancer stem cells from breast cancer transplantation tumors in nude mice.Methods Human breast cancer MDA-MB-231 cells were injected into the right axilla subcutaneous of 20 nude mice,and the tumor growth was observed.After 30 days,tumors were isolated and stained with hematoxylin and eosin,and then tumor cells from tissues were isolated.DMEM medium containing serum was used to cultivate isolated transplantation tumor cells,cell morphology and growth were also observed.Flow cytometry was used to detect the proportion of stem cells(CD44^(+)/CD24^(-/low) cells)in transplantation tumor cells.Serum-free DMEM medium containing epidermal growth factor(EGF),basic fibroblast growth factor(bFGF)and B27 cell supplement were used to cultivate transplantation tumor cells and to obtain cell microspheres.The proportion of stem cells on the 10th day in cell microspheres was detected by using flow cell sorter and stem cells were isolated according to the markers of cell surface.Results After subcutaneously injecting MDA-MB-231 cells into 20 nude mice for 9 days,17 nude mice had subcutaneous tumors with more parenchymal cells,little interstitial cells,arranged cords tumor cells,large volume of the cell and abundant cytoplasm,the nuclei in different sizes and hyperchromatic state,mitotic more common,the nucleoli clear and obvious pleomorphy.After cultivating transplantation tumor cells with DMEM medium containing serum,the cells began to grow adherent after 24 h,and the adherent proportion rose to 60% after 3 days;after 7 days,the cell proliferation was accelerated;and the cell morphology was more consistent,most of which were spindle shaped and were not significantly different from MDA-MB-231 cells;the proportion of stem cells in transplantation tumor cells was(0.10±0.02)%.After cultivating transplantation tumor cells with serum-free DMEM medium containing cell cultured supplement,the cells grow in spherical patterns,the proportion of stem cells in cell microspheres got up to(70.47±2.03)%
作者
李新涛
胡珍
李朱斌
耿倩倩
Li Xintao;Hu Zhen;Li Zhubin;Geng Qianqian(Department of Internal Medicine 2,Shaanxi Provincial Cancer Hospital,Xi'an 710061,China;Department of Gynaecology,Xi'an Maternal and Child Health Hospital,Xi'an 710002,China;Department of Minimally Invasive Intervention,Shaanxi Provincial Cancer Hospital,Xi'an 710061,China;Department of Nuclear Medicine,the First Affiliated Hospital of Xi'an Jiaotong University,Xi'an 710061,China)
出处
《肿瘤研究与临床》
CAS
2022年第10期721-725,共5页
Cancer Research and Clinic
基金
国家自然科学基金(81803015)。
关键词
乳腺肿瘤
肿瘤干细胞
肿瘤移植
乳房肿瘤
实验性
细胞分离
小鼠
裸
流式细胞术
Breast neoplasms
Neoplastic stem cells
Neoplasm transplantation
Mammary neoplasms,experimental
Cell separation
Mice,nude
Flow cytometry
