| Objective :1. To selection cancer stem cell-like subgroup from heterogeneous subpopulation cells in human laryngeal carcinoma.2. Comparison of the differences of cell surface marker expression on laryngeal cancer stem cell-like cell subsets and different subsets, to initial screen cell surface markers of laryngeal cancer stem cell.Method:1. Laryngeal carcinoma cells were cultured in vitro by primary tissue culture technique.The samples were taken from the 15cases diagnosed as squamous cell carcinoma Department of Otolaryngology ,the First Affiliated Hospital of Shanxi Medical University.2. Heterogeneous subpopulations were isolated by limiting dilution. To select two clones having the biggest differences in morphology from subpopulations named type 1 and type 2 subsets.Type 1 and type 2 subsets were continued to single cell subcloned cell culture. Comparison of differences in morphology and differentiation .3.To detect the proliferative activity of type 1 and type 2 subsets by MTT and the cell cycle of two subsets by flow cytometry; each subpopulation was implanted in nude mice subcutaneously to measure the tumor forming ability.4.Immunocytochemical staining technology and flow cytometry were used to detect the expression of the putative stem cell marker CD133,CD44 in Type 1 and Type 2 subsets cells.5. All data were expressed as mean±standard deviation, and analyzed by T-test with SPSS16.0. P <0.05was considered as significant statistically difference.Results:1. Laryngeal carcinoma cells was isolated from type 1 and type 2 cell morphology between the two largest sub-group. 1 spindle cell growth, fewer processes, training did not result in cloned offspring cell; 2 cells into polygonal, multi-processes, to continue to foster offspring produced by cloning cells in the presence of heterogeneity.2. MTT cells detected two types of value-added, sub-group of type 2 cells 5 days after inoculation, showed more than one type of cell the value-added activity. 3. The relative DNA content by flow cytometry to determine the cell percentage of each phase. Most of type 2 cells in G0/G1 phase was significantly higher than type 1 cells were significantly different by the t test (P <0.05).4.1-type tumor cells grown subcutaneously in nude mice after 6 weeks no tumor formation (0 / 5); 2 tumor cells implanted subcutaneously in nude mice 2 weeks after the formation of tumors, tumor was 100% (5 / 5). 2 subsets polygonal cell progeny cells grown subcloned into nude mice, tumor formation rate of 100% (5 / 5); and 1 spindle cell progeny planted to nude mice no tumor. Value-added capability in vitro, in the G0/G1 cell cycle was significantly higher than the proportion of type 1 cells, is a cancer stem cell characteristics of subsets.5. The immunofluorescence staining showed that cells of type 1 subsets CD44-FITC, CD133-FITC staining was negative; 2 sub-population cells stained strongly positive for CD44-FITC, covered with climbing film, CD133-FITC staining constant positive, and type 2 cells with a certain amount, are expressed in cells surrounding the green fluorescence intensity.6. Flow cytometry shows CD44, CD133 subsets in type 1 cells did not express; 2 subset cells, 95.1%±2.3% of the cells expressed membrane antigens CD44, 23.8%±1.7% of the cells expressed membrane antigen CD133. And cellular immunofluorescence technique results in good agreement.Conclusion:1. Primary laryngeal carcinoma cells are heterogeneous with respect to characteristics on morphology,proliferation,functional and tumor forming ability in vitro. Type 2 cells possess subsets have biological characteristics of cancer stem cells.2. Membrane antigen CD133, CD44 can be used as the surface of laryngeal cancer stem cell markers.
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