The Effects Of Infrasound Therapy On Raji Cell And Peripheral Blood Lymphocytes Of Human In Vitro

Infrasound is a mechanical vibration wave with frequency between 0.0001～20Hz. Experts from many countries have regarded the potential use of infrasound in clinical diagnosis and treatment increasingly. There were few reports from abroad about the assistant treatment on pain by infrasound therapy. No literature of research on patient with cancer by infrasound therapy was found yet. The influence of infrasound on tumor was at the stage of researching. Even there were few literatures about infrasound in vitro abroad; there was no internal report of research on tumor cells and lymphocytes treated by infrasound. And that such researches are necessary for the security of infrasound therapy in patients with tumor. The information about the biological effect of infrasound result mostly from the experiments with infrasound source from nature or infrasound cabin. The research on the'clinical security equipment'of infrasound therapy and its physic parameter was quite few, and that was the basis of developing and using infrasound therapy in clinical and laboratory.Some of our previous researches about the biological effect of infrasound have been done. During the 'Experimental study on the protecting effect of Jia Wei Bu Yang Huan Wu Decoction to damage caused by infrasound', the traditional Chinese herbs were used to try to protect the damage caused by infrasound. BALB/c mice were randomized into normal control groups, infrasound control groups, infrasound and drug-treated groups. Learning and memory ability, oxidation resistance in the brain (content of MDA, activity of GSH—Px and activity of SOD as yardsticks) and ultra-structure of pallium were observed after 14 days. The results showed that infrasound can bring damages to mice, which mainly lies in decline of learning and memory ability, changes of activity of SOD and GSH-Px, content of MDA in the body increasing, the change of ultra-structure in pallium. Jia Wei Bu Yang Huan Wu Decoction treated by therapy can improve learning and memory ability by promoting the activity of SOD and GSH—Px, eliminating excessive free radicals, lowering lipid per oxidation to decrease the content of MDA and lighten the damnification of pallium. Different dose has different protection effects.We also used INFRASOUND 8? , a therapeutic infrasound generator, to treat the vitro cultured HL-60 cells (treatment groups). The cells of control groups were exposed to air. The proliferation of the cells would be measured respectively at 15min., 30min., 60min., 90min. and 120min. after the treatment. The cell viability would be showed by trypan blue staining, the cell multiplication measured by MTT assay, and the apoptosis of the cells detected with flow cytometry. Results showed that the differences of the cell proliferations in all experimental conditions were not significant.In this research, the physical parameters of infrasound-field generated by a therapeutic infrasound emitting device would be measured (that would be the basis of the next experiment), and the effects of infrasound therapy on Raji cell and peripheral blood lymphocytes of human in vitro be studied. The purpose was to provide the experimental references for infrasound therapy on patients with tumor and the study on the influence on the lymphocyte of human.1. Study on the characteristics of infrasound-field generated by a therapeutic infrasound-emitting device1.1 Material and methods: INFRASOUND 8? was used as a therapeutic infrasound generator. The infrasound signal was measured by 'Infrasound signal data collecting and analyzing system' developed by the 4th Military Medical University. During measuring, the sensor of the system was put on less than 1cm from the center of the applicator of the infrasound generator. The collecting of the infrasound signal would be started after the generator had been working for 5min.. The test would be carried on separately as the generator worked in 3 modes (selection buttons). Measurement would be continued for 60s once a time. The collecting frequencies of the infrasound signal included 20Hz, 16Hz, 12Hz, 8Hz, and 4Hz. The sound signals under 20Hz collected for 15s noiselessly would be intercepted from it each 0.2s interval. Then the parameters included frequency, percentage of infrasound and non-infrasound, dB of infrasound, and other information would be recorded and analyzed.1.2 Results: The characteristics of infrasound-field generated by a therapeutic infrasound emitting device showed that while the therapeutic infrasound generator was operating, both the infrasound levels and frequency range (4, 8, 12,16 and 20Hz) were increased with the selective buttons, but the infrasound energy from all selection buttons was less than 90dB. The sound signals above 20Hz were produced too with infrasound-field when the device was working. The signals of main frequency of infrasound were chaotic. That means the infrasound signals of all frequencies generated by INFRASOUND 8? could be in the time of the highest or lest intensity during working.1.3 Conclusions: The characteristics of infrasound-field generated by the therapeutic infrasound generator might be significant to prevent the damage caused by over bio-resonance during infrasound therapy. The different characteristics of infrasound-field might act differently on human body in clinical.2. Influence of Infrasound on Multiplication of Raji Cell2.1 Material and methods: INFRASOUND 8? was used as a therapeutic infrasound generator. Raji cell line was cultivated commonly in RPMI-1640 culture medium containing 10% fetus cattle serum. Infrasound group: the applicator of the infrasound generator was put on 1.5～2.0cm from the surface of liquid in the dish contained Raji cells. Infrasound generator worked at selection button 3. Control group: treated same as infrasound group but the generator was powered off. Raji cells would be treated for 15,30,60,90,min.separately, and tested with trypan blue assay, MTT assay, flow cytometry analysis (Annexin V-EGFP / PI), and scanning electron microscope after Oh., 24h. and 48h. cultivations. The experiment would be repeated for 3 times.2.2 ResultsTrypan blue assay: The numbers of survival cells treated by infrasound for 120min. after 24h. cultivation were slight less than that of its control group, but the difference was no statistic significant (P>0.05). Infrasound groups (treated for 90min., 120min. and then cultivated for 48h.) were ditto. The rest groups had no significant differences in them (P>0.05).MTT assay: Even OD counts of all infrasound groups seemed less than their control groups; but the differences were no significant (P>0.05).Flow cytometry analysis: Percentages of survival cells in all groups were more than 90%, that of necrotic cells and apoptosis cells were less than 10%; and that the differences in all groups were not obvious (P>0.05).SEM: The cells treated by infrasound exposure for 120min. and then cultivated for 24h. showed that the prominent or micro-floss of the membrane become shorten and decreased. The surface of the membrane became smooth. But the figure of the cells was intact. There was a small nub on the membrane in one or two cells that could be the 'apoptosis nub'.2.3 Conclusions: Infrasound treatment in the experiment had no obvious influence on multiplication and apoptosis of Raji cells. But the membrane of Raji cell would be affected directly by infrasound, and its penetration could be changed.3. Effect of Infrasound on Human Peripheral Blood Lymphocytes2.1 Material and methods: Samples of human peripheral blood lymphocytes were taken from 18 health volunteers and each sample was tripartite into 3 groups (n=18 for each group): infrasound group (treated by infrasound generator), control group (treated same as infrasound group but the generator was in power off), and bank group (extracted lymphocytes from human blood were cultivated in incubated tank with 5%CO2 and saturated humidity all through). All samples would be experimental treated for 15, 30, 90, and 120min. and then cultivated for 24h and 48h. separately. Trypan blue assay, MTT assay, flow cytometry analysis (Annexin V-EGFP / PI), and scanning electron microscope would be used for the test.2.2 ResultsThe results of trypan blue assay showed that the rates of survival lymphocytes in all groups were above 95%.MTT assay: OD counts of lymphocytes in infrasound groups were no differences from their control groups (P>0.05), but that of the infrasound groups showed increasing with the time of infrasound treatment. OD counts of lymphocytes in control groups did not display such a tendency.Flow cytometry analysis: Percentages of apoptosis lymphocytes and other cells had no significant difference in all groups (P>0.05).SEM: The lymphocytes treated by infrasound exposure for 120min. and then cultivated for 48h. showed that the prominent or micro-floss of the membrane and the figure were almost same as the lymphocytes of blank group. But the lymphocytes of control group showed that the prominent or micro-floss of the membrane become shorten or decreased, the abnormal figure of the lymphocytes increased.3.3 Conclusions: Infrasound treatment in the experiment might increase the active of human peripheral blood lymphocytes, but the influence on apoptosis of lymphocytes was not obvious. The membrane of lymphocytes would be affected directly by infrasound, and its penetration could be changed. Infrasound therapy might prevent natural aging of lymphocytes and damages from environment. That might be related with the mechanisms of infrasound effect on lymphocytes.4. SummaryCharacteristics of infrasound-field generated by INFRASOUND 8? : While the therapeutic infrasound generator was operating, both the infrasound levels and frequency range (4, 8, 12,16 and 20Hz) were increased with the selective buttons, but the infrasound energy from all selection buttons was less than 90dB. The sound signals above 20Hz were produced too with infrasound-field when the device was working. The signals of main frequency of infrasound were chaotic. Infrasound used in our research was bellow 20Hz and more than 69% of the sound signals.Infrasound treatment in the experiment had no obvious influence on multiplication and apoptosis of Raji cells. But the membrane of Raji cell would be affected directly by a quite long infrasound treatment. Infrasound treatment in the experiment might increase the active of human peripheral blood lymphocytes, but the influence on apoptosis of lymphocytes was not obvious. The membrane of lymphocytes would be affected directly by infrasound. Infrasound therapy might prevent natural aging of lymphocytes and damages from environment.The biological effects of INFRASOUND 8^TMtreatment on Raji cells and lymphocytes might be related with the changes of cell membrane. It should be explored further that whether the selectivity of infrasound influence on normal and abnormal cells were existed.