| Radiotherapy has been focused on external beam radiation, and externally applied radiation is prejudicial for sensitive components from normal cells. Therefore, scientists have been exploring methods to improve radiotherapy of tumors while to reduce radiation damage to the surrounding normal tissue. In 1946, Na 131I was used for the treatment of thyroid cancer for the first time. It's over 60 years that the negative electron irradiation in vivo was applied to treat cancer. As for the biological effects of ionizing radiation, both the positron electron and negative electron can kill tumor cells. In recent years, researches on tumor therapy by positron are gradually carried out. Currently in clinics, 18F–fluorodeoxyglucose (18F-2-Deoxy-2-Fluoro- D-Glucose, 18F-FDG) as the representa- tive of radiopharmaceutical emitting positron is being widely used in PET imaging. 18Fluorine is a positron emitter with a short physical half life and a range of 0.1-0.2 millimeter in tissue. Secondary gamma radiation after positron-electron annihilation is may be detected by PET, but there have been suggestions that the preferential uptake of 18F-FDG by tumors could be exploited in a therapeutic setting. There is a high incidence of liver cancer in China. Recently, incidence and mortality of liver cancer has been increasing slowly and its mortality rank third in all cancers. Although there are several methods to treat liver cancer, it's one of the worst prognoses in a variety of solid tumors. The five-year survival rate of patients with liver cancer is only 14%-30%. Thus, studies must be carried out for new approaches to treat liver cancer. Because 18F-FDG can be taken up preferentially by primary liver cancer and its metastases, the higher malignancy of tumor, the more exuberant cellular metabolism, the higher 18F-FDG uptake rate, the better treatment effects. So, 18F-FDG can be most plausible candidate to be used in targeted treatment of liver cancer.Objectives: Morphological changes, radiation injury-related indicators and the expression apoptosis-related gene were observed to investigate the influence of 18F-FDG on the apoptosis of HepG2 cells and elucidate possible mechanism of the apoptosis induced by 18F-FDG.This may provide experimental basis for the use of radiopharmaceuticals emitting positron brachytherapy in primary and metastatic foci of liver cancer in the future.Methods: (1) Cell culture: HepG2 cells were grown in DMEM medium containing 10% FBS and 100U/ml penicillin and streptomycin (complete medium)at 37℃in 5%CO2. Subculture once per three days; (2) Cell irradiation: At 24h after the passage, HepG2 cells at the logarithm growth stage were treated by 18F-FDG for 24h and then were used in the following experiments, whose final radioactive concentration was 0, 0.925×107Bq/mL, 9.25×107Bq/mL, 92.5×107Bq/mL, respectively. (3) Cell morphology observation: Cells were observed under general inverted microscope for morphology and pictures were taken by digital camera.(4) Determination of cell proliferation by MTT method: Various groups of cells were trypsinized into single-cell suspension by 0.25% trypsin, the cell density is adjusted as 1×105/ml, inoculated in 96-well plates with100ul per hole, added 100ul complete medium per hole, cultured for 24h. 18F-FDG was added into experimental groups while the same volume DMEM medium was added into blank groups. After 6, 12, 24 h, per hole adding 20ul 5mg/ml MTT solution. Continue to foster for 4h then abandon it. Per hole add DMSO 150ul and shock for 10min. Afer detecting optical density value in each hole using the microplate reader (at wavelength 570nm), calculating the inhibitory rate of cell proliferation. 5) The apoptosis rate: Cultured cells were digested by 0.125% trypsin and made into single cell suspension with culture medium. Living cells were counted and cell concentration was adjusted to 5×105/ml. After centrifuging, cells were collected and washed with PBS. Then cells were fixed by 70% ethanol precooled at–20℃for1h and washed with PBS again. After incubating with 5mg/ml RNase at 37℃for 30min, cells were dyed by 10μg/ml Propidium iodide (PI) solution. Cells were ready for test after 15min reaction in the dark. Apoptotic cell rate were analyzed by flow cytometry . (6)The determination of hydrogen peroxide (H2O2) in the cell: HepG2 cells during logarithmic growth phase were treated for 24h and then were washed twice with PBS, incubated with 150μl of 20μmol/L DCFH-DA work solution at 37℃in CO2 incubator for 30 min, washed thrice with PBS, fluorescence intensity was measured by flow cytometry at excitation wavelength 485 nm and emission wavelength 538nm, 10000 cells were counted for each sample, WinMDI software was used to analyze mean fluorescence intensity (MFI).(7)The determination of gene expression by reverse transcription polymerase chain reaction (RT-PCR):First, extracted the total RNA according to the instructions of Trizol total RNA extraction kit. The total RNA were extracted for RT-PCR. Then, the products were electrophoresized in gel at 100V, observed under gel imager and photographed. Each band was semi-quantitatively analyzed by Bandscan software. Their gray value divided by the one ofβ-actin was named as Relative intensity(RI). The changes of gene expression were observed through the RI. (8)Statistic analysis: Measured data were expressed as?x±s. SPSS (10.0) software was used for statistical processing. The analysis of variance of single factor and t test were performed. Linear regression was used to analyze dependency between variables.Results: (1) Typical apoptotic cells were observed under the optical microscope in per 18F-FDG treated group. The cell volume become smaller, cytoplasm condensed, small nuclei increased and pyknosis. The density of apoptotic cells increased with the increase of 18F-FDG radioactive concentration. (2) After 6, 12 and 24 h, the proliferation of cells treated by 18F-FDG were inhibited. Inhibition rate increased with the increment of 18F-FDG radioactive concentration and the time .(3) With the increase of 18F-FDG radioactive concentration, cell apoptosis rate increased gradually. Apoptosis rate (%) of treated HepG2 cell were 3.3±0.01,4.5±0.07,16.9±0.23 (each group compare to the apoptotic rate of control group 0.5±0.31, P <0.05) (4) HepG2 cells were treated with different radioactive concentrations of 18F-FDG for 6 hours. the content of H2O2 in cells in irradiated group was more than that in control(P<0.01), and the content of H2O2 in cells increased with the augmentation of 18F-FDG radioactive concentration. (5) HepG2 cells were treated with different radioactive concentrations of 18F-FDG for 24 hours, the expression of P53 gene and rad51 gene increased.Conclusion: 18F-FDG irradiation could induce HepG2 cells apoptosis, and the apoptosis rate increased with. the increment of its radioactive concentration. |
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