A way to enrich the rat hepatic cancer stem cells

Objective To establish a novel approach of enriching rat hepatic cancer stem cells (HCSCs) in vitro and identify the biological features of HCSCs.Methods Based on the previously established thee-step method for stem cells isolation,HCSCs were enriched briefly as follows.Firstly the rat liver cancer model was made by injecting diethylnitrosamine (DENA) intraperitoneally.By using modified Selgen's two-step method,the rat liver cancer cells were isolated.After these cells were cultured for 10 days,the first step was done to purify the cells with differential trypsinization and differential adherence.When the cells spread more than 90％ pavement of culture flask,the second step was done to fractionate the cells with Percoll continuous gradient centrifugation.To identify the properties of stem cells from each fraction,the third step was done,included detection of three markers of HCSCs [ epithelial cell adhesion molecule (EpCAM),CD133,alpha fetal protein (AFP)] by using flow cytometry and reverse transcription-polymerase chain reaction ( RT-PCR),and assay of the multiplication capacity and invasion of cells from every fraction.Results The hepatic cancer cells at the fraction 3 were most likely enriched with HCSCs,which had the highest proliferative ability.The matrigel invasion assay indicated that the largest number (92.750 ±4.432) of cells at the fraction 3 migrated through the membrane,which was significantly different from the others.Flow cytometry revealed that the expression of EpCAM ( 84.50 ± 8.31 ) ％ and AFP (85.20 ±8.31 )％ at fraction 3 was significantly different from the others.RT-PCR revealed that the expression of EpCAM ( 111.34 ± 5.59 ) and AFP ( 13.76 ± 0.95 ) at the fraction 3 was significantly different from the others.The expression of CD133 at the fraction 3 ( 17.16 ±0.42) was significantly different from that at the fraction 2 or fraction 4,but insignificantly different from that at the fraction 1.Conclusion A simple and practical method for enriching HCSCs in vitro was establisbed,which may supply a new approach of harvesting cells for the study on HCSCs targeted therapy for hepatocellular carcinoma. Key words: Carcinoma, hepatocellular; Stem cell; Cell isolation