| Background: Osteosarcoma is a common high degree of malignancy and mortality of tumors, which can occur early lung metastasis and have in a peak incidence in children and adolescents ages[1-2]. In current, early surgery to clear the primary lesion, combined with chemotherapy drugs after surgery, is the major treatment, but the osteosarcoma cells aren't completely eradicated, while the patients with postoperative recurrence or metastasis lesions are still so many that the present treatment study of osteosarcoma lost in trouble. Nowadays, the advances of science and technology make the interdisciplinary fusion research development become a new model, while domestic and foreign scholars have tried to combine physics means with biological methods to study the malignant tumor. Extremely low frequency electromagnetic field's( ELF-EMF) effects on malignant tumor, in recent years, is a new model of the interdisciplinary fusion researches, and has achieved certain achievements. Current researches have confirmed that certain parameters of ELF-EMF can inhibit the proliferation or induce apoptosis of malignant tumor cells[3-8]. Moreover, early in 1979, the American FDA had officially approved ELF-EMF can be used in the treatment and rehabilitation of clinical disease, which certificated that appropriate ELF-EMF's effects on the human body was safe and reliable. In recent years, the rise of cancer stem cell theory has also provided a new idea and direction to break the predicament for the treatment of malignant bone tumors. The theory is that the growth and proliferation of tumor tissue depends on a few cancer stem cells, when most other cells have not or only limited proliferative capacity, and only this part of the stem cells can be immortalized. Only effectively inhibit or kill the cancer stem cells can bring a major breakthrough for the cancer treatment and research. It was proved that side population cells of cancer(SP cells) had stem cell's characteristics[9-10]. Therefore, this experiment selected the osteosarcoma HOS cells as the research object. Firstly, by contrasting the assay of ELF-EMF exposure group's proliferation, apoptosis, colony formation and invasion with the control group, different intensities and frequencies of ELF-EMF's biological effects on the osteosarcoma HOS cells will be filtered, and then we could select the optimal parameter of ELF-EMF, with a comprehensive evaluation, in vitro. Then, by using the optimized parameters of ELF-EMF to irradiate the osteosarcoma HOS cells,we will observe the SP cells ratio changes with the control group. By the flow cytometry, we select SP cells to culture routinely. Using the optimized parameter of ELF-EMF to irradiate SP cells and changing intervention conditions, with the western blot analyzing the targeted protein's expression, we will study the possible signaling pathways mechanisms. We hope that this series of experiments and researches could provide some experimental basis and mechanisms, for clinically applying ELF-EMF to treat the osteosarcoma, finally.1. Biological effects and filtering the optimal parameter of the ELF-EMF on the osteosarcoma HOS cells in vitroObjective:By comparing the assay of ELF-EMF group's proliferation, apoptosis, colony formation and invasion with the control group, we would screen different intensities and frequencies of ELF-EMF's biological effects on the HOS cells, and choose the best parameter of ELF-EMF, with the statistical analysis, in vitro. Methods: Conventional culture HOS cells were irradiated two hours per one day, five days of continuous irradiation, in vitro, with different magnetic field frequencies( 1 Hz, 5 Hz, 10 Hz, 15 Hz, 20 Hz, 30 Hz, 40 Hz, 50 Hz), which were fixed with magnetic field intensities( 0.1 m T, 1 m T, 5 m T, 10 m T, 15 m T), comparing with the control groups. Then we could calculate the HOS cells' proliferation and inhibition rate between different groups, with the CCK-8 assay, according to the OD. After statistically analyze, two stronger inhibition of ELF-EMF parameter on the HOS cells were filtered, which were validated by the assay of apoptosis, colony formation and invasion again. Results: Through statistical analysis of each group's OD value, we arrived at the conclusion that the inhibition rate of( 1 Hz, 0.1 m T) group was the most,( 40 Hz, 10 m T) group followed(P <0.05). The result of the flow cytometry showed that( 40 Hz, 10 m T) group's apoptosis ratio was higher than the other two groups(P <0.05). Colony formati on assay showed that the most efficiency of clone formation was the control group,( 1 Hz, 0.1 m T) group was the second, and( 40 Hz, 10 m T) group was the least(p<0.05). In the invasion assay, the number of( 40 Hz, 10 m T) group's HOS cells through Transwell chambers was significantly lower than the other two groups(P <0.05). Conclusion: The osteosarcoma HOS cells can be significantly inhibited to proliferate, reduced to clone, and slowed down to invase, apoptosis of which can also be induced, by the( 40 Hz, 10 m T) group of the ELF-EMF parameter.2. A mechanism study of the ELF-EMF' effects on the osteosarcoma HOS cells in vitroObjective:The side population cells'( SP cells) ratios of the osteosarcoma HOS cells were observed between the experimental group and the control group, which were irradiated by the optimal parameter of ELF-EMF. And then the possible role of signaling pathways mechanisms were studied by adding the cisplatin( CDDP) that could up-regulate VEGF/Flt1 self secretion signal into the medium, with the assay of western blot. Methods: SP cells' ratios of HOS cells were identified, by the flow cytometry, between the ELF-EMF irradiation group and the control group. SP cells,which were filtered by the flow cytometry, were divided into the optimal parameter of ELF-EMF irradiation group, the CDDP co-culture group, the CDDP co-culture plus ELF-EMF irradiation group, and the control group, while the targeted protein's expression was tested by the western blot assay. Results: After the flow cytometry's double staining analysis, SP cell ratio of the optimal parameter of ELF-EMF irradiation group was lower than the control group, and the difference was statistically significant(P <0.05). The results of western blot showed that: SP cells' VEGF/Flt1 protein expression of the optimal parameter of ELF-EMF irradiation group significantly decreased, when the phospho-ERK1,2 phosphorylation of SP cells were significantly inhibited, compared with the control group, but the expression of ERK1,2 had no significant variation in groups. Conclusion: The optimal parameter of ELF-EMF irradiation may inhibit the proliferation of the osteosarcoma HOS cells, finally, by down-regulating the VEGF/Flt1 protein expression or inhibiting the phospho-ERK1,2 phosphorylation of SP cells. |
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