摘要
该文主要探索从小鼠Lewis肺癌亲代细胞系(Lewis lung carcinoma parental cell line,LLC-Parental)中分离能够在体外长期培养的肿瘤干细胞(cancer stem cells,CSCs)的方法。在体外贴壁条件下培养LLC-Parental细胞,通过连续富集悬浮生长的细胞,经过8代后,成功富集出可悬浮成球的小鼠Lewis肺癌干细胞样细胞株(LLC-spheroid enrichment,LLC-SE)。为了验证其干细胞特性,我们使用荧光定量PCR法(Real-time PCR)检测表示干细胞特性的基因Bmi1(B-cell-specifi c moloney murine leukemia virus insertion site 1)、Cd133(cluster of differentiation-133)、Aldh1a1(aldehyde dehydrogenase family 1,subfamily A1)、Oct4(octamer-binding transcription factor 4)、Sox2(sexdetermining region Y box 2)、Nanog和Klf4(Kruppel-like factor 4)的表达。6孔板琼脂成球实验检测其增殖和悬浮成球能力。96孔板单细胞克隆形成实验检测单个细胞的克隆形成能力。裸鼠皮下移植这一干细胞特性检测的经典模型检测其体内致瘤能力以及C57同源小鼠左肺原位种植模型检测其肿瘤转移能力。Real-time PCR结果显示,LLC-SE细胞中Bmi1的表达水平显著高于LLC-Parental细胞。6孔板琼脂成球实验、96孔板单细胞克隆形成实验表明,LLC-SE细胞的悬浮成球能力和单细胞克隆能力显著高于LLC-Parental细胞。动物实验表明,LLC-SE细胞的体内致瘤能力、肿瘤转移能力强于LLC-Parental细胞。该文首次运用连续8次悬浮聚球法分离获得大量的可稳定传代的具有干细胞特性的LLC-SE细胞,为建立肿瘤干细胞细胞模型提供了新的方法,为进一步研究肿瘤干细胞的生物学特性奠定了基础。
We explored a new method for isolation and identification of cancer stem cells（CSCs） which could be passaged continuously from parental Lewis lung carcinoma cells（LLC-Parental）. The LLC-spheroid enrichment（LLC-SE） culture can be established through 8 consecutive rounds of collecting floating cells that become independent of attachment for growth and form spheroids from the LLC-Parental cells. For stem cell properties verification, the expressions of pluripotent genes Bmi1（B-cell-specific moloney murine leukemia virus insertion site 1）, Cd133（cluster of differentiation-133）, Aldh1a1（aldehyde dehydrogenase family 1, subfamily A1）, Oct4（octamer-binding transcription factor 4）, Sox2（sex-determining region Y box 2）, Nanog and Klf4（Kruppellike factor 4） were measured by Real-time PCR. The abilities of proliferation and spheroid formation were assessed by 6-wells plate agar spheroid formation assay. The ability of single cell cloning was analyzed by 96-wells plate single cell cloning assay. In vivo tumorigenicity was measured by nude mice subcutaneous transplantation which was considered as the classical model for stem cell property assessment. And tumor metastasis ability was measured by left lung orthotopic implanting in syngeneic C57 mice. The results of Real-time PCR showed that the expression of Bmi1 in LLC-SE was much higher than that in LLC-Parental. The results of spheroid formation assay and cloning assay showed that spheroid formation ability and single cell cloning ability of LLC-SE were better than that of LLC-Parental. The results of in vivo experiments showed that tumorigenicity and metastasis ability of LLC-SE were better than that of LLC-Parental. In this work, we developed a new method for the isolation of longlasting CSCs from cultured tumor cells. This method will allow the generation of cellular models for mechanistic characterization of CSCs.
作者
刘永利
孔亮盛
孙志卫
王健宇
邢若曦
Liu Yongli Kong Liangsheng Sun Zhiwei Wang Jianyu Xing Ruoxi(Laboratory of Translational Cancer Stem Cell Research, Institute of Life Sciences, Chongqing Medical University, Chongqing 400016, China)
出处
《中国细胞生物学学报》
CAS
CSCD
2017年第1期35-43,共9页
Chinese Journal of Cell Biology
基金
国家自然科学基金(批准号:81272405)
重庆市教委2015年度科学技术研究项目(批准号:KJ1500222)资助的课题~~
