有限稀释法分选人骨肉瘤143-B干细胞及鉴定

A method of limited dilution to sort human osteosarcoma 143-B stem cells and its identification

目的:探索人骨肉瘤143-B干细胞的分选及鉴定方法。方法:采用有限稀释法对143-B细胞进行干细胞的分选,并检测所获取细胞表面干细胞标志物Nanog和OCT4的表达水平;对所获取的细胞进行成骨及成脂诱导分化,以检测其是否具有多向分化的潜能;动物成瘤实验鉴定细胞的成瘤能力,耐药实验检测顺铂对细胞半数抑制浓度(half inhibitory concentration,IC50)的影响。结果:分选获得的单克隆143-L细胞具有较高的成球能力;143-L细胞表面Nanog和OCT4 m RNA和蛋白的表达水平均明显高于亲代143-B细胞;对其进行成骨及成脂多向诱导分化成功。143-L细胞的成瘤能力及耐药能力(顺铂对143-B及143-L细胞的IC50值分别为0.83和2.51μg/m L)均明显高于143-B细胞。结论:有限稀释法可以用于143-B细胞干细胞的分选。

Objective：To explore the methods of cell sorting and indentification of human osteosarcoma 143-B stem cells.Methods：The 143-B cells were treated by limited dilution method for stem cell sorting,and the expression levels of Nanog and 0CT4 were determined.The multipotentials of osteogenic and adipogenic differentiation were detected.The ablility of tumorigenesis was examined by tumor formation experiment in vivo,and the drug resistance assay was performed to detect the effect of cisplatin on half inhibitory concentration（IC_（50））.Results：The monoclonal 143-L cells obtained by cell sorting had a high ability of sphere formation.The Nanog and 0CT4 expression levels on obtained monoclonal cells（143-L cells） were significantly higher than those in 143-B cells.Meanwhile,the 143-B cells had multipotentials of osteogenic,adipogenic and chondrogenic differentiation.Furthermore,the tumorigenicity in nude mice and drug resistant ability of143-L cells（the IC_（50）values of 143-B and 143-L cells were 0.83 and 2.51μg/mL,respectively）were stronger than those of 143-B cells.Conclusion：The limited dilution method can be used to sort 143-B stem cells.