| Objective The theory of cancer stem cells (CSCs) offer us a new thought about tumors, more and more kinds of cancer stem cells were isolated and identified form corresponding tissue. Our aim was to isolate and identify cancer spheres in pancreatic cancer cell lines PANC-1 and ASPC-1 with serum-free medium (SFM) including epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), insulin and B27 supplement, and to detect the expression level of miR-590-3p by real-time Q-PCR (Qrt-PCR).Methods Differentiation assay, self-renewal assay, cell cycle, cell invasion assay, xenograft experiment and the expression of the surface markers CD24/CD44 were performed to characterize CSCs in PANC-1 and ASPC-1 spheres culturing with serum-free medium, respectively. Moreover, qRT-PCR was used to detect the expression of miR-590-3p.Results A minority subpopulation of cells in both ASPC-1 and PANC-1 survived, passaged and formed spheres in the absence of serum. Cell spheres differentiation occurred when serum was supplemented in SFM, G0/G1 stage in ASPC-1 and PANC-1 cell spheres were (75.3±5.4) %,(80.1±4.7)%, respectively. The mean of migrated cells of PANC-1 and ASPC-1 spheres was 147.3±18.6 and 113.2±12.9 that stated the spheres have higher invasive ability compared with those of parental cells. As few as 5×10~5 cells in PANC-1 and ASPC-1 spheres could initiate xenograft tumor in NOD/SCID mice, which suggested the tumorigenicity of spheres was 100 times that of parental cell lines. The percentages of CD24+CD44+ in spheres were 0.38%-0.43% and 4.91%-5.21%, which showed increased expression compared with ASPC-1 and PANC-1 cell lines (P<0.05). The expression of miR-590-3p in spheres was 4.67, 4.52 times its expression in cell lines, respectively. (P<0.05).Conclusions Pancreatic cancer cell spheres may be a source for collecting CSCs by culturing with serum-free medium. miR-590-3p maybe play an important role in regulating biological characteristics of pancreatic cancer stem cells. |
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