采用罗丹明123对肝癌细胞系MHCC97(Rholow)干细胞亚群的分离及鉴定

目的 建立分离肝癌细胞系MHCC97细胞中对罗丹明123染料具有不同排斥能力细胞亚群的方法,并探讨其在肝癌干细胞和异质性研究中的意义.方法 利用流式细胞分选术(FCM)并结合不同浓度的罗丹明123(Rho123)染料,分析和分选肝癌细胞系MHCC97细胞中具有不同染料排斥能力的肝癌细胞亚群,再通过细胞生长的测定、软琼脂克隆形成以及免疫组织化学检测和裸鼠成瘤实验等方法,以比较不同肝癌细胞亚群的差异.结果 根据不同染料排斥能力可以将肝癌细胞系MHCC97细胞分为Rholow和Rhohigh2个亚群,2个亚群在增殖能力、克隆形成率以及甲胎蛋白(AFP)和角蛋白-19(CK-19)表达以及裸鼠成瘤实验上差异有统计学意义,Rholow亚群具有干细胞特性.结论 罗丹明123结合FCM可以富集具有干细胞特性的Rholow亚群,同时为进一步揭示肝癌异质性机制奠定基础.

Isolation and identification of heterogenic subpopulation from MHCC97 cell lines of human hepatocellular carcinoma(HCC)by using Rhodamine123 extrusion assay

Objective To establish an approach of dissociating different subpopulation in MHCC97 cell lines of human hepatocellulKr carcinoma(HCC)according to the ability of these ccHs to extrude the Rho123 dye and discuss its role in the investigation of cancer stem cells and heterogeneity.Methods Flow cytometry in combination with different concentrations of Rho123 was used to analyze and sort subpopulation according to the abihty of these cells to extrude the Rho123 dye.Viable cell count,soft agar cloning method,immunocytochemical staining and tumorigenicity investigation were performed to show the heterogeneity in Rholow and Rhohigh subpopulation.Resuits Rholow cells and Rhohigh cells were sorted using flow cytometry according to the ability of these cells to extrude the Rho123 dye.There was significant difference in growth rate,soft agar cloning,expression of alpha-fetoproteins(AFP)and Cytokeratin 19 (CK-19),tumorigenicity investigation between Rho123low and Rho123high subpopulation.Conclusion Subpopulation with stem cell properties in MHCC97 cell lines can be inriched by Rho123/FCM,indicating HCC is heterogeneous further.