| Objective:To isolate,culture and identify of CD44~+CD133~+laryngeal cancer stem cells from laryngeal carcinoma Hep-2 cell line and observe its biological characteristics in vitro.Study of CD44~+CD133~+laryngeal carcinoma Hep-2 cell phenotype of stem cell proliferation and growth,invasion,adhesion ability,colony formation ability and resistance and a series of characteristics of stem cells.To do some new research on the theory of laryngeal cancer stem cells and possible target therapy.Methods:Containing 10%FBS DMEM culture medium liquid cultured human laryngeal carcinoma Hep-2 cells,magnetic activated cell sorting CD44~+CD133~+,CD44~+CD133~-,CD44~-CD133~+,CD44~-CD133~-four subsets,was measured by CCK-8 kit of separation of Hep-2 cells and four sub types determination of proliferation,of 5 kinds of cell growth curve was drawn by flow cytometry sorting detection positive rate,Transwell chamber invasion assay and adhesion assay to evaluate the invasion and adhesion ability of clone formation.Experimental evaluation of the single cell differentiation potential.Under the effect of cisplatin detection of the resistance.Results:1.The expression rate of each cell was detected by flow cytometry.The expression rates of CD44~+CD133~+,CD44~+CD133~-,CD44~-CD133~+and CD44~-CD133~-subset were89.62%,4.04%,5.16%,1.18%,respectively.2.The biological characteristics of 5 kinds of cells in Hep-2,CD44~+CD133~+,CD44~+CD133~-,CD44~-CD133~+and CD44~-CD133~-were analyzed.The ability of proliferation,invasion,adhesion,clone formation and drug resistance of CD44~+CD133~+cell subsets were higher than that of Hep-2 cells.CD44~+CD133~+>CD44~-CD133~+>Hep2>CD44~+CD133~->CD44~-CD133~-Conclusion:MACS is an effective method for sorting CD44+CD133+cells.CD44+is an important factor in the maintenance of stem cell characteristics of laryngeal cancer stem cells.CD44+CD133+cell subsets have obvious characteristics of cancer stem cells.and can make some new exploration for the high expression markers of laryngeal cancer cells. |
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