| Objective：Observing the morphological characteristics and the radiation resistance of the foursubsets in the breast cancer cell line MCF-7, understanding whether the cell subsets ofCD44+/CD24-/lowpossesses the properties of stem cell.Methods：The single cell suspension of MCF-7cells in logarithmic growth phase were preparedby adding of CD44-FITC and of CD24-PE antibody, sorted by flow cytometry to get thefour groups including CD44+/CD24-/lowsubsets,CD44+/CD24+cell subsets, CD44-/CD24+cellsubsets, CD44-/CD24-cell subsets, four groups of cells sortied by flow cytometry wereinoculated to contain poly-lysine coated coverslide in the culture dish, in the scanningelectron microscope, observated the cell morphology and photographed. Then these fourgroups of cells were resuspended in DMEM containing Matrigel (1:1, V/V),1ml syringe ofthe above cell suspension was injected into the front side of the armpit of the randomized5week old female NOD/SCID mice (about22g), the mice were killed by neck breaking andtumor growth was observed after12weeks. After adding of CD44-FITC, and5ul ofCD24-PE antibodies, MCF-7cell suspension is rinsed twice with PBS, adding5ul DAPIMatrigel plastic coated.After the same time, by a laser scanning confocal microscope camera(400×), counting the number of cells in MCF-7cell lines in various subsets of cells throughMatrigel biological glue down man holes and collected three-dimensional images tocalculate the migration distance. And then single cell suspension is prepared in logarithmicgrowth phase MCF-7cells, adjusting cell density to3×105/mL and vaccinated to six wellplate2ml per hole, until the adherent cells for given with0Gy,2Gy,4Gy,6Gy,8Gy,10Gyirradiation. Cells were collected after48h, the percentage of apoptotic cell subsets by flowcytometry. The single cell suspension is prepared into in logarithmic growth phase MCF-7cells, adjust ed cell density to3×105/mL, were given with0Gy,2Gy,6Gy,8Gy doseirradiation,48h after irradiation10ul of CD44-FITC and5ul of CD24-PE antibody wereadded, using a laser scanning confocal microscope camera (400×), counting all subsets ofcells in the MCF-7cell lines the number of cells through Matrigel biological glue drop holesand collect three-dimensional images to calculate the migration distance. The single cell suspension were prepared into in logarithmic growth phase MCF-7cells, adjusted celldensity to3×105/mL vaccination to six well plate2ml per hole, until the adherent cells for0Gy2Gy,6Gy exposure, were collected at12h,24h,36h,48h,72h cells, preparation ofsingle cell suspension and adjusting cell concentration5×105/100ul, were added to10ul ofCD44-FITC and5ul of CD24-PE and of CFSE (working concentration of10ug/ul), thenusing the various cell subsets by flow cytometry fluorescence ratio calculation of the variouscell subsetsResult：This study displays in MCF-7cell by the scanning electron microscopy: the surface ofCD44-/CD24+subsets is protruding less,surrounded without pseudopodia. The cell surfaceof CD44+/CD24+subsets is protrusionsmore, the surrounding can be seen a small numberof pseudopodia, the cell surface of CD44-/CD24-subsets is smooth, protruding less andfewer pseudopodia, the cell surface of CD44+/CD24-/lowsubsets is protruding microvilli andpseudo-feet, than the other three groups of cells are more pseudopodia than theCD44+/CD24+subsets; we also found in MCF-7cells the CD44-/CD24+subsets andCD44-/CD24-subsets reinfusion1×105cells was not observed in tumor formation whenCD44+/CD24+subsets is injected with1×105cells of CD44+/CD24+subsets of the visibletumor(1/6). The CD44+/CD24-/lowsubsets were injected cell count which greater than1×103to tumorigenicity, and the tumorigenicity time is shorter, a larger proportion of tumorformation, the size of formated tumor is larger; in Transwell experiments we found that inthe CD44+/CD24-/lowsubsets migratory cell number and migration distance is1to3timesthan the other three groups of cells,and stronger than the other three groups of cells, itsinvasion of the strongest may be associated with tumor metastasis and invasion. In thisstudy, MCF-7cells the various cell subsets after2Gy irradiation is compared tounirradiated cell the apoptosis rate is overall improvement, but the proportion of apoptosisis no significant increase by flow cytometry. In irradiation4Gy or6Gy, the apoptosis ratewas significantly increased,8or10Gy irradiation, there is a higher rate of apoptosis, but thedifference was obvious. With the increasing of radiation dose, apoptosis rate theCD44+/CD24-/lowsubsets compared to other three groups of cells, the apoptosis rate of theminimum, equivalent to about1/3-1/6of the other three groups of cells at the same dose;byTranswell experiments,the study displayed the MCF-7cells after2Gy irradiation differentcell subsets were compared with unirradiated,the cell number is increased with the doseincreasing, the number of cells through the polycarbonate cool film gradually reduced when dose reach to6Gy, the cell count is less than unirradiated through the the polycarbonatecool film, migration of cells was significantly reduced when the dose reached8Gy. Butwhether irradiated or not, the CD44+/CD24-/lowsubsets compared with the other cell subsetsof cells through the polycarbonate cool film is the largest. Unirradiated cell subsets throughthe cell membrane cell count gap, in2Gy irradiation, the biggest gap of CD44+/CD24-/lowsubsets through polycarbonate cool cell numbers is equivalent to approximately the othercell populations2-9times, with the increasing of radiation dose, to reach6Gy or8Gy, thegap gradually decreases; CFSE fluorescence intensity is related with cell division and decay,by calculating the speed of the decreasing efficiency of the fluorescence intensity in all cellwe could subsetsthe proliferation of various cell subsets in the evaluation of MCF-7cellscell lines. The results showed that when unirradiated, joined CFSE12h after ofCD44+/CD24-/lowcell subsets fluorescence incorporation of8294,equivalent toapproximately4-6times the other three groups of cells, cultured to72h, the cell subsetsThe fluorescence intensity is close. But when exposured the2Gy and joined CFSE at12hof CD44+/CD24-/lowcell fluorescence incorporation is14589, equivalent to approximately3-6times of the other three groups of cells, cultured to72h. the various cell subsets influorescence intensity is close. CFSE absorption rate of the cell subsets increasedfluorescence intensity. The relut shows that the fluorescence decay of the intensity of thesubsets of CD44+/CD24-/lowcell is the fastest compared with the other three groups of cells,the cell division is rapidest.Conclusion:1.The electron microscope images can be seen of CD44+/CD24-/lowsubsets of microvilliprotruding foot a lot, with strong characteristics of the migratory ability.2. In MCF-7cell populations, CD44+/CD24-/lowsubsets tumorigenicity is strongest.3. The CD44+/CD24-/lowsubsets has strong ability of invasion and metastasis, not onlywith radiation resistance, but also with stem cell invasion and metastasis, indicating that inMCF-7cell lines of CD44+/CD24-/lowsubsets with radiation resistance.4. The CD44+/CD24-/lowsubsets with stem cell properties, radiation resistance bydifferent doses of irradiation, the CD44+/CD24-/lowsubsets showed strong anti-apoptotic andproliferative capacity in maintaining the stability of groups and plays an important role. |
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