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OBJECTIVE: Tumor stem cells present in the tumor tissue with self-renewal and differentiation potential of cells, tumor initiating cells maintain tumor growth, the root cause of tumor recurrence and metastasis. Sorting identification of cancer stem cells can make us a clearer understanding of its characteristics, in order to find new targeted drugs to treat cancer, improve the cure rate. High expression of CD55 (CD55hig) and low CD55 expression (CD55low) cells in breast cancer MCF-7 cells was determined by flow cytometry mean fluorescence intensity the sorting CD55hig and CD55low cells, cell morphology was observed adherence rate, colony formation and cell cycle distribution, to explore that CD55hig whether the cells have cancer stem cell characteristics. Isolated from a breast cancer stem cells in order to find new methods and theoretical basis for the study of breast cancer stem cells. Methods: complete medium DMEM containing 10% fetal bovine serum, cultured breast cancer MCF-7 cell line, take trypsin digestion logarithmic phase cells into single cell suspension, the first set of added nucleic acid dyes by Hoechst33342. The cancer stem cell surface expression of specific ATP-binding cassette (ATPbinding cassette, ABC), also known as the ABC transporter protein ABC transporter protein efflux Hoechst33342 role stem cells can be lightly stained. The second group joined the nucleic acid dye Hoechst33342 staining Verapami as a control group. Verapami can block the function of the ABC transporter protein in cancer stem cell surface. Side population (side population, SP) cells and the main group (mainpopulation, MP) cell ratio and separation of flow cytometry SP stem cells and MP subsets stem cells CD55 monoclonal antibody marker SP and MP subpopulations CD55 control antibody labeled SP stem cells and MP subpopulation cells as a negative control, determination of SP stem cells and MP subpopulations of CD55 mean fluorescence intensity, comparing the differences of the two confirmed SP the stem cells CD55hig cells, CD55 monoclonal antibody markers not sorting MCF-7 cells measured CD55hig cells and CD55low cell proportion, and sorting to collect CD55hig cells the class stem cells and CD55low cells detect biological characteristics: 1. determination of adherence rates: CD55hig stem cells and CD55low cells were seeded in culture plates, each well 104, containing 10% fetal bovine serum DMEM complete medium culture after 6h, 12h, 18h and 24h counting adherent cells, cell adherence rate (%) = adherent cell number / seeded cells × 100%; determination of colony formation rate, counting the number of clones: take for 200 CD55hig stem cells and CD55low cells seeded in 10cm petri dishes, DMEM complete medium to visible clone weeks. Colony formation rate (%) = number of clones / inoculation cell number × 100%; Cell cycle analysis: CD55hig stem cells, CD55low cells and unsorted MCF-7 cells were PI dye, and analyzed by flow cytometry after incubation The cell cycle distribution. Results: 1.MCF-7 cells joined Hoechst33342 dyes, flow cytometry to select the highest point of the active cell density analysis, the proportion of stem cells in the MCF-7 SP 4.34 ± 0.59%, this ratio was significantly reduced presence of verapamil 0.1 ± 0.07%, 33.66 ± 7.89% MP cell proportion; 2. collect SP stem cells and MP cell marker CD55 monoclonal antibody measurement was SP stem cells CD55 in average fluorescence density of 100.85 ± 4.57, MP sub-group of cells CD55 of The mean fluorescence intensity was 50.51 ± 4.75. CD55 monoclonal antibody labeled MCF-7 cells, measured CD55hig proportion of stem cells was 2.12 ± 0.39% the CD55low cell ratio was 20.10 ± 5.29%; 3.CD55hig stem cells and CD55low cells were seeded in 24-well cell culture plate, CD55low cells adherence was 40.87 ± 1.55% 6h, 12h adherent was 55.40 ± 2.46%, the 18h adherent was 85.10 ± 3.36%. The CD55hig class stem cell 6h stickers wall was 29.53 ± 1.20% the 12h adherent was 46.40 ± 2.99%, the 18h adherent rate of 77.2 ± 1.07%, the CD55low cells adherent was significantly higher than CD55hig class stem cells, P lt; 0.05 significant difference the 24h when CD55low cells adherent was 96.3 ± 0.96%, CD55hig kind of stem cell adherence was 95.5 ± 1.1%, P gt; 0.05, no statistical difference; observed under the microscope after inoculation, 12h after CD55hig class of stem cells are mostly spherical CD55low cells adherent fusiform; 4.CD55hig stem cells and CD55low cells, colony forming ability, CD55hig class of stem cells cultured for one week cloned formation rate was 20.04 ± 1.07%, lower than the CD55low subpopulations 27.14 ± 1.07%, P lt; 0.05, significant difference; 5. flow analysis found CD55hig stem cells in G0/G1 quiescent cells accounted for 85.4 ± 3.37%, significantly higher than CD55low cells (58.6 ± 2.55 %) and MCF-7 cells (70.73 ± 4.21%), P lt; 0.05, statistically significant. CD55hig class of stem cells in the proliferation of cells in S phase accounted for only 13.93 ± 3.45%, significantly lower than CD55low cells (40.36 ± 2.56%) and MCF-7 cells (24.93 ± 2.86%), P lt; 0.05, statistically significant . Conclusion: 1. Labeled nucleic acid dye Hoechst33342 breast cancer MCF-7 cell line, flow cytometry lightly stained SP cells, this part of the cells was significantly reduced after verapamil, blocking, MCF-7 cell lines contain SP cells, SP cells cancer stem cells; 2.CD55 monoclonal antibody labeled elected SP stem cells and MP cells were detected in the SP stem cells CD55 mean fluorescence intensity the fluorescence density value is higher than that of the MP cells, this value is detected CD55 monoclonal antibody labeled undivided elect MCF-7 cells, found in the small number of CD55hig cells; 3.CD55hig cells most quiescent, non-adherent in suspension and cloning hereditary CD55hig cell biological characteristics of cancer stem cells.
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