Research On Cell Culture System With Extremely Low-frequency Alternating Magnetic Field And Its Biological Effects

Cancer is a serious threat to human health and is one of the most common causes of death in the world.Although the existing anti-tumor therapy has made some progress,the standard anti-tumor therapy based on chemotherapy drugs and radiotherapy still meets potential side effects.In recent years,some studies have shown that extremely low-frequency and low intensity magnetic fields are harmless to normal cells,and may even be beneficial,while some malignant tumors will respond to this kind of magnetic fields.With the advantages of small side effects,low cost,wide application and noninvasive,extremely low-frequency magnetic fields(<300Hz,ELF-MFs)have been confirmed that ELF-MFs are involved in the regulation of tumor cell cycle distribution,apoptosis,autophagy,differentiation,systemic immunity and the inhibition of angiogenesis and metastasis through a variety of signaling pathways.In addition,in the combination therapy with chemotherapy drugs,ELF-MFs can not only promote the absorption of drugs by stimulating small holes on the surface of tumor cells,but also enhance the effect of chemotherapy drugs and reduce the dose of chemotherapy drugs by regulating cell cycle and apoptosis related proteins.However,although there are many studies on the biological effects of ELF-MFs on tumor cells,because of the differences in the applied magnetic field types,magnetic field parameters and tumor cell types,the research results in this field are not consistent and repeatable.To solve these problems,in order to explore the potential application value of ELFMFs in the field of tumor treatment,this paper carried out the research on cell culture system and biological effects of extremely low-frequency alternating magnetic field.The main research contents of this article are as follows:(1)Cell culture system with extremely low-frequency alternating magnetic field exposure environment was developed.In order to explore the effects of extremely lowfrequency alternating magnetic field on tumor cells,we should not only consider the basic conditions of cell culture,but also produce a uniform magnetic field irradiation environment in the space enough to cover the cell culture vessel.In this paper,we proposed a magnetic field built-in cell culture system based on the existing commercial cell culture incubator.After improving the ordinary Helmholtz coils,coils are placed in the cell culture incubator.A series resonant circuit based on H-bridge is used to drive the coil to generate extremely low-frequency alternating magnetic field with controllable intensity and frequency.In order to incorporate the exposure direction of magnetic fields into the experimental design,we proposed to arrange large size 3D Helmholtz coils outside of the cell culture incubator.In order to avoid the influence of conventional metal cell incubator on the distribution of magnetic fields,acrylic was selected as the material of cell incubator,and the cell incubator was embedded in the three-dimensional Helmholtz coils according to the size of the uniform area generated by the coils,so as to realize the application of extremely low-frequency alternating magnetic field with controllable intensity,frequency and direction.(2)In view of the poor repeatability and consistency of the effects of extremely low-frequency magnetic fields on tumor cells caused by the differences in magnetic field type,intensity,frequency,treatment duration and samples settings,we analyzed the effects of extremely low frequency alternating magnetic field on cell proliferation from four aspects,namely,magnetic field irradiation direction,field strength,frequency and cell type.With the system in which magnetic fields are generated outside of the cell incubator,we designed control experiments of cell survival rate.We concluded that the effects of perpendicular exposure to cell culture plane on cell proliferation was more significant than that of parallel irradiation.With the magnetic fields built-in cell culture system,the effects of extremely low-frequency alternating magnetic field intensity and frequency on cell proliferation were tested.The survival rate of tumor cells gradually decreased with the increase of intensity of ELF-MFs.The response of different kinds of tumor cells to magnetic field frequency is different.The inhibitory effects of extremely low-frequency alternating magnetic field on common epithelial cells are limited.(3)In view of the unclear mechanism of cell biological effects caused by extremely low-frequency alternating magnetic field,we used TMT-labeled proteomics and mass spectrometry to analyze the spectrograms of breast cancer cell MCF-7 with/without the irradiation in extremely low-frequency magnetic field(200Hz,1m T).The differentially expressed proteins were screened by bioinformatics analysis methods.The Gene Ontology(GO)and Kyoto Encyclopedia of Genes and Genomes database(KEGG)were used to analyze,annotate,localize and enrich the differentially expressed proteins to explore the mechanism involved.The preliminary experimental verification was carried out by combining Western blotting and flow cytometry,which provided guidance for the follow-up study on the mechanism of extremely low frequency alternating magnetic field inhibiting tumor cell proliferation based on proteomics.(4)In order to explore the mechanisms of apoptosis induced by extremely low frequency alternating magnetic field in breast cancer cells and explain how extremely low-frequency alternating magnetic field inhibits the proliferation of tumor cells,we designed western blot,flow cytometry,fluorescence detection experiments to study the mechanisms involved in the apoptosis and cell cycle arrest triggered by extremely lowfrequency alternating magnetic fields.We found that the phenomenon of cell cycle arrest and apoptosis increased with the exposure time,and the increase of cells at G2-M phase is related to Cyclin B1 reduction induced by extremely low-frequency alternating magnetic fields.The occurrence of apoptosis is related to the generation of reactive oxygen species induced by extremely low-frequency alternating magnetic fields,the inhibition of PI3K/AKT signaling pathway,and the activation of GSK-3β.We repeated experiments with GSK-3β inhibitors to confirm the key role of GSK-3β in the induction of apoptosis by extremely low-frequency alternating magnetic field,which could provide theoretical basis for the phenomenon of extremely low-frequency alternating magnetic field inhibiting the proliferation of tumor cells.Through the study of the above contents,the innovations of this paper are as follows:(1)In order to solve the problems of device design and mechanism research in biological effects of extremely low-frequency alternating magnetic field,cell culture systems of ELF alternating magnetic fields were developed.We tested the effects of irradiation direction,intensity and frequency of extremely low-frequency alternating magnetic field on the proliferation of different cell lines were systematically studied.We used cell survival rate detection to test the effects of changes in parameters to cell proliferation.It was verified that the inhibition effect of vertical irradiation magnetic fields on the proliferation of tumor cells was obvious,and the cell inhibitory response was dependent on the magnetic field intensity.The response of different types of tumor cell lines to frequency was different,while magnetic field exposure showed no significant effects on the proliferation of normal epithelial cells.(2)In view of the unclear mechanism of biological effects caused by extremely low-frequency magnetic fields,we analyzed the TMT-labeled proteomics of breast cancer cells irradiated by extremely low-frequency alternating magnetic field.With the help of mass spectrometry and bioinformatics analysis methods,by comparing the spectrograms of MCF-7 cells irradiated by extremely low frequency alternating magnetic field for 24 hours with that of untreated MCF-7 cells,the differentially expressed proteins were screened out and analyzed for localization,annotation and enrichment.The potential mechanism of biological effects of ELF alternating magnetic field on breast cancer cells was revealed,which provided a basis for further study of extremely low-frequency alternating magnetic field.Besides,we provided a new idea for the biological effect of tumor cells induced by extremely low-frequency magnetic field.(3)The mechanism involved in the induction of apoptosis and regulation of cell cycle distribution by extremely low-frequency alternating magnetic fields was studied.We designed western blot,flow cytometry,fluorescence detection and reactive oxygen species detection tests to explore the mechanism of cell cycle arrest and apoptosis induced by extremely low-frequency alternating magnetic fields.It is suggested that the decrease of cyclin B1 was responsible for the cells aggregation at G2-M phase which was induced by extremely low-frequency alternating magnetic field,while cell apoptosis is related to the generation of reactive oxygen species and the activation of GSK-3β induced by extremely low-frequency alternating magnetic field,which could provide a theoretical basis for the phenomenon that extremely low frequency alternating magnetic field inhibits cell proliferation.