Effects Of Low Frequency Pulsed Electromagnetic Fields On Cardiac Microvascular Endothelial Cells

Background and purpose of the experimentIn recent years, the role of the magnetic field has been paid gradually attention, it is a safe and effective means of physical therapy in the treatment of some diseases such as bone and joints and it has achieved good effect.Some researches have already showed that with the application of electromagnetic fields (EMFs) human umbilical vein endothelial cells and bovine aortic endothelial cells culture in vitro in blood vessel formation can be effectively promoted, FGF-2 secretion can also be promoted by the magnetic field, so people are constantly looking for a non-invasive therapy using magnetic field to improve cardiovascular disease. In recent years, the incidence of ischemic heart disease increased year by year, the treatment of ischemic heart disease is to promote endothelial regeneration and angiogenesis. Microvascular endothelial cells(CMECs) have many functions similar to endothelial cells , they can participate in the regeneration of blood vessels, they are an important component of the heart, and played an important part in improve cardiovascular disease . But now few studies were reported in this area, and we can only move forward in the exploration to find biological effects of magnetic field to play the best waveform, intensity and time, so that in the treatment of cardiovascular diseases took another step forward for its in lay in the foundation for the clinical application.MethodsPart 1 Effects of different waveforms of pulsed electromagnetic fields on myocardial microvascular endothelial cells1.Using trypsin and collagenase digestion method isolated cardiac microvascular endothelial cells , with differential adhesion method purified adherent cells.2. The cells were randomly divided into two sections: the rectangular and the triangular waveform group,each large group was divided into control and treatment groups ,and each treatment group were divided into four magnetic fields intensity,they are 0.6mT, 1.2mT, 1.8mT and 2.4mT .Control group did not receive magnetic field exposure, and other conditions of control group were consistent with the treatment group. The treatment groups were passaged and then cultured in the incubator for a period of time, when cell adhesion were observed we start to radiat. The frequency of pulsed magnetic field is 15Hz, irradiation time was 4h / d, continuous exposure 5d. CCK-8 was used to detected cell proliferation,cell migration was detected by transwell, the flow cytometry assayed cell cycle changes, FITC labeled phalloidin staining of the cytoskeleton and NO secretion was measured by Griess assay.Part 2 Effects of triangular waveform pulsed magnetic field on proliferation, migration and cell cycle of myocardial microvascular endothelial cell 1. Using trypsin and collagenase digestion method isolated cardiac microvascular endothelial cells , with differential adhesion method purified adherent cells.2. Cardiac microvascular endothelial cells were divided into four groups: control group, 1.0mT group, 1.4mT group and 1.8mT group. Control group did not receive magnetic field exposure, and other conditions of control group were consistent with the treatment group. When cell adhesion were observed we start to radiat. The frequency of pulsed magnetic field is 15Hz, irradiation time was 4h / d, continuous exposure 3d. MTT was used to detected cell proliferation,cell migration was detected by transwell, the flow cytometry assayed cell cycle changes. ResultsPart 1 Effects of different waveforms of pulsed electromagnetic fields on myocardial microvascular endothelial cells1. Successfully cultured cardiac microvascular endothelial cells and successfully purified.2. CCK-8 detected that after exposuring to the rectangular waveform magnetic field the levels of cell proliferation had raised compared with the control group, the most significant proliferation group is 1.8mT group (0.9679±0.057 vs 0.7552±0.0334; P <0.01 ).Triangle waveform magnetic filed significantly accelerated the proliferation of 0.6mT group and 1.8mT group compared with the control group (P <0.05) ,but 1.2mT group and 2.4mT group compared with the control group showed no significant difference (P> 0.05).3. Rectangular waveform magnetic field accelerated the migration of CMECs in each group (pre/view) .Compared with the control group the migrated number of CMECs significantly increased, in which the 1.8mT group was the most effective (48.00±4.18 vs 23.80±3.11, P <0.01).Compared with the control group, triangle waveform magnetic filed accelerated the migration of 0.6mT group and 1.8mT group significantly (P <0.05), compared with the control group 1.2mT group and 2.4mT group did not significantly increased (P> 0.05).4. By flow cytometry, cells irradiated by rectangular waveform magnetic field compared with the control group cells G1 phase, S phase and G2 phase were significant changes, (G2 + S) phase cells were increased ( P <0.01), the most significant change group is 1.8mT group.Triangle waveform magnetic filed of 1.8mT group and 0.6mT group had some effects on the cell cycle (P <0.05), the cell cycle of 1.2mT group and 2.4mT compared with the control group is almost no change (P> 0.05).5. With the fluorescence microscope, rectangular waveform magnetic field reduce the rate of apoptosis in each group (P <0.05). Triangle waveform magnetic filed of 1.8mT group and 0.6mT group had similar effect in reduce the apoptosis rate of CMECs(P <0.05), The apoptosis rate of 1.2mT group and 2.4mT group show no significant difference compared with the control group (2.18±0.19 vs 2.30±0.35; 2.24±0.27 vs 2.30±0.35; P> 0.05).6. After expose to the rectangular waveform magnetic field cells the cytoskeletal structure of the CMECs reorganized compared with control group , Immunofluorescent staining of each group showed that the stress of fiber membrane increased, the changes of 0.6mT group and 1.8mT group is the most significant, Triangle waveform magnetic filed of 0.6mmT group and 1.8mT group also has the same performance.7. After 5d, NO secretion tests showed that compared with the control group, NO secretion in each group were significantly increased (P <0.01).Triangle waveform magnetic filed of 0.6mT group and 1.8mT group compared with the control group were also accelerated (P <0.05), 1.2mT group and 2.4mT showed no significant increase on NO secretion compared with the control group (P>0.05).Part 2 Effects of triangular waveform pulsed magnetic field on proliferation, migration and cell cycle of myocardial microvascular endothelial cell1. MTT colorimetric assay showed that, the proliferation of 1.4mT group and 1.8mT group of CMECs were significantly increased compared with the control group (P <0.05); No obvious difference were detected in the 1.0mT group of proliferation(P>0.05).2. Transwell assaied the migration of CMECs, cells were treated with crystal violet staining. The statistical analysis shows that the magnetic field can promote the migration of the cells. The migration of 1.4mT group and 1.8mT group was significantly different compared with the control group (P <0.05), 1.0mT group and the control group showed no significant difference (P> 0.05).3. Cell cycle by flow cytometry showed that cells exposed to 3 days, The G1 phase, S phase and G2phase of 1.4mT group and 1.8mT was statistically difference, cell proliferation and differentiation increased significantly. The change of 1.8mT group is the most significant (P <0.05).ConclusionAt a fixed frequency, the biological effects of rat cardiac microvascular endothelial cell are closely related to the intension and the waveforms of pulsed magnetic fields.