efficient enrichment of hepatic cancer stem-like cells from a primary rat hcc model via a density gradient centrifugation-centered method高效浓缩的肝癌症干细胞样细胞主要通过密度梯度centrifugation-centered鼠肝癌模型的方法.pdfVIP

Efficient Enrichment of Hepatic Cancer Stem-Like Cells from a Primary Rat HCC Model via a Density Gradient Centrifugation-Centered Method 1,2. 2. 2. 2. 1,2 1 2 Wei-hui Liu , Xing Wang , Nan You , Kai-shan Tao , Tao Wang , Li-jun Tang *, Ke-feng Dou * 1 PLA Center of General Surgery, General Hospital of Chengdu Army Region, Chengdu, Sichuan Province, China, 2 Department of Hepatobiliary Surgery, Xijing Hospital, Fourth Military Medical University, Xi’an, Shaanxi Province, China Abstract Background: Because few definitive markers are available for hepatic cancer stem cells (HCSCs), based on physical rather than immunochemical properties, we applied a novel method to enrich HCSCs. Methodology: After hepatic tumor cells (HTCs) were first isolated from diethylinitrosamine-induced F344 rat HCC model using percoll discontinuous gradient centrifugation (PDGC) and purified via differential trypsinization and differential attachment (DTDA), they were separated into four fractions using percoll continuous gradient centrifugation (PCGC) and sequentially designated as fractions I–IV (FI–IV). Morphological characteristics, mRNA and protein levels of stem cell markers, proliferative abilities, induced differentiation, in vitro migratory capacities, in vitro chemo-resistant capacities, and in vivo malignant capacities were determined for the cells of each fraction. Findings: As the density of cells increased, 22.18%, 11.62%, 4.73% and 61.47% of primary cultured HTCs were segregated in FI–FIV, respectively. The cells from FIII (density between 1.041 and 1.062 g/ml) displayed a higher nuclear-cytoplasmic ratio and fewer organelles and expressed higher levels of stem cell marke