大鼠肝癌肺转移灶中边缘群细胞的分离与表型鉴定
Isolation and identification of side populations from lung metastatic hepa-tocellular carcinoma in rats
张佳斌 1窦科峰 1李韧 1刘正才 1宋文杰 1千年松 1于恒超1
作者信息
- 1. 第四军医大学附属西京医院肝胆外科,陕西,西安,710033
- 折叠
摘要
目的:建立肝癌远处器官转移灶中边缘群肝癌细胞的分离方法并对其干细胞特征进行鉴定.方法:利用二乙基亚硝胺(DEN)建立大鼠肝癌肺转移灶模型;组织块原代培养法扩增培养肺转移灶中的肝癌细胞,对细胞进行Ho-echst33342及不同荧光标记的c-kit,Sca-1 mAb染色后,利用流式细胞仪(FACS)分选肝癌肺转移灶中的边缘群及非边缘群细胞,并分析两群细胞表面干细胞标志物的表达特征;软琼脂克隆形成分析两群细胞的肿瘤形成能力.结果:成功建立大鼠肝癌肺转移灶模型,并在此基础上获得性质较为均一并可稳定传代的肺转移肝癌细胞株;FACS分析表明边缘群细胞占肺转移肝癌细胞总细胞量的0.5%,边缘群细胞表面c-kit表达率为85%,Sca-1表达率为75%,而非边缘群细胞表面c-kit及Sca-1表达率分别仅为8%和3%;软琼脂克隆实验结果表明边缘群细胞平均克隆形成率为69.9%,非边缘群细胞平均克隆形成率为15.3%,边缘群细胞肿瘤形成能力显著高于非边缘群细胞(P<0.01).结论:肺转移肝癌细胞中的边缘群细胞具备显著的肿瘤干细胞特征.
Abstract
AIM: To establish the methods of isolating and identifying the stem-cell-like hepatocytes from rat pulmonary metastatic hepatocellular carcinoma. METHODS: Rat model of hepatocellular carcinoma with pulmonary metastasis was established by diethylinitrosamine. Hepatocellular carcinoma cells from pulmonary metastasis were proliferated by tissue culture. Side population (SP) cells and non-side population cells were isolated from the cultured cells by Hoechst33342 staining with FACS and were followed by identification for the expression of tumor stem cell markers c-kit and Sca-1 with FACS. Soft agar clone formation assay was performed to determine the tumorigenesis capacity of SP and non-SP cells from hepatocarcinoma cells. RESULTS: The rat model of hepatocellular carcinoma with pulmonary metastasis was established and stable hepatocellular carcinoma cell line was harvested from the model. Side population cells accounted for 0.5% of the total pulmonary metastatic cells. c-kit and Sca-1 were highly expressed on SP cells, with the expression rate of 85% and 75% respectively in SP cells, and 8% and 3% respectively in non-SP cells. Soft agar clone formation rates were 69.90% and 15.25% respectively in SP and non-SP cells, showing that the tumorigenesis capacity of SP cells was higher than that of non-SP cells (P ＜ 0.01). CONCLUSION: SP pulmonary metastasis hepatocellular carcinoma cells display the features of tumor stem cells.
关键词
癌,肝细胞/肿瘤转移/边缘群/肿瘤干细胞Key words
caicinoma, hepatocellular/neoplasm metastasis/side population/tumor stem cells引用本文复制引用
出版年
2009
NETL
NSTL
维普
万方数据