| PurposeTo analyze whether the radiosensitizing effect of taxotere on human nasopharyngeal carcinoma cell line ( CNE - 1) in vitro is related to the changes of its cellular cycle and to observe whether it can induce apoptosis of CNE - 1.Materials and methods1. Cell CultureThe human nasopharyngeal carcinoma cell line, CNE - 1, was provided by the Virus Institute of Chinese Preventive Medicine Academy of Science. CNE - 1 cells were grown in PRMI 1640 medium (GIBCO BRL, USA) supplemented with 10% bovine calf serum and maintained in a 5% CO2 atmosphere at 37℃ with 100% humidity. Cells were grown as monolayers in flasks and those in exponential growth phase were selected into use.2. Drug Treatment and IrradiationTaxotere was obtained from AVENTIS Co. and diluted to the desired concentrations (5 ×10-8 mg/ml) in PRMI 1640 medium. Cellswere irradiation with X - ray emitting from SIEMENS Linear accelerator at a dose rate of 200μy/min. The source skin distance is 100cm and the irradiation field is 40 × 40cm.3. Flow CytometryExponentially growing cells were trypsinized, counted for the live ones and planted into culture flasks. And 12 hours later, remove the medium.Medium (4ml) was added into the control and the simple irradiation ones and taxotere - containing medium (5 × 10 -8 mg/ml, 4ml) was added into the simple taxotere and the combination of taxotere with irradiation ones. The medium was aspirated out 12 hr later and the cells were rinsed twice with PBS. Then fresh medium (4ml) without taxotere was added into each one.An immediately irradiation was given to the simple irradiation and the combination taxotere with irradiation ones. The radiation dose was 2 Gy.At the incubation times of 0,6,12,18,24,36 and 48hr, the cells were harvested after trypsinized, washed twice, resuspended in PBS and fixed with 70% ice - cold ethanol at 4℃ for at least half an hour. The ethanol was removed before flow cytometric analysis and 0. 1% Triton X - 100μ/ml),10mg/ml RNase (200μl), 50μg/ml PI (100μl) were added in order for 10, 45, 60min and at room temperature, 37℃, and 4℃ respectively. The cells were stained under no light.The stained cells were measured on a FACScan flow cytometer with CELLQuest software and the percentage of cells in each phase was calculated using ModFit 2. 0 software. Flow studies measured at least 1 ×104 cells.4. ImmunohistochemistryTwo cover glasses were put into each petri dish with the diameter of 6cm. Exponentially growing cells were trypsinized, counted for the live ones and planted onto the cover glasses in definite cell number. About half an hour later with anchoring incubating, medium (4ml) was added into each petri dish and then another 12hr incubating was given. After that remove the medium.Medium (4ml) was added into the control ones and taxotere -containing medium (5 ×10-8 mg/ml, 4ml) was added into the taxotere ones. The medium was aspirated out 12 hr later and the cells were rinsed twice with PBS. Then fresh medium (4ml) without taxotere was added into each one. The cover glasses were taken out 48 hrs later and were rinsed three times with PBS. Then they were fixed with 95% ethanol for 10min and were rinsed three times with PBS.This time the cover glasses were dipped inside 0. 1% Triton X -100 liquid for 20min and then immunohistochemistry staining was given in order to the direction of the Immunohistochemistry Test Kit.Streptavidin -Peroxidase (S - P) Method was used and the primary antibodies were immediately usable. Both of the primary antibodies used in this study and the S - P Test Kit were products of Maxim Biotechnology Development Company. One was a rabbit against human polyclonal antibody for Caspase - Pan and the other was a mouse against human monoclonal antibody for bcl -2.5. Data and imagesCharts were drawn with SPSS 10.0 software and the image results of immunohistochemistry were taken with photomicroscope.ResultsCompared with the control group, a G2 + M block was present with exposure...
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