AFM Studies About The Effects Of Magnetic Field And/or Anticancer Drug On Cancer Cell At Cellular And Subcellular Level

Aim:The combination effect of satic magneitc field and anti-cancer drug on the morphology of cancer cells was observed at the cells and subcellular level. AFM observation system on cells in atmosphere and physiology solution was established.AFM was used to observe the changes on the surface of different cancer cells which was delt with the cancer drug and magnetic field.As the research went on,the research transferred from cellular level to subcellular level. The effect of magnetic field and cancer drug on DNA which was extracted from cancer cells was observed.The mechanism about the effects of magnetic field and/or anticancer drug on cells was investigated.Method:AFM was used to observe the surface of cells which were fixed by different kinds of fixing agents. The suitable fixing agent which can preserve the fine structure of the cellular surface well was choosed,and the concentration of fixing agent was various for different cells. The quality of the image which was decided by the different parameters of AFM, such as scanning mode and scanning frequency, was evaluated. The sample preparation condition and the observation parameters were optimized. The fine structure on the surface of the suspension cells which was fixed with glutaraldehyde was observed, and the fine structures of the surface of the adherent cells were observed at the atmosphere or the physiology solution environments, respectively. DNA was extracted from the cells irradiated by UV or the magnetic field combined with cisplatin, and the molecule structure of DNA was observed by AFM. DNA in "comet" was observed in situ by the enzyme digest method.Result:1. The essential experiment parameters about observing cells at the atmosphere and the physiology solution environment have been gained. Different kinds of fixed cells and living cells were observed by this experiment system. The fixed cells such as K562 cell (chronic myelogenous leukemia cell), SW480 cell (colorectal adenocarcinoma cell), the mouse hepatocyte, Hepa 1-6 cell (hepatoma cell of mouse) and the human erythrocyte were observed at the atmosphere environment. And the SMMC-7721 cell (human hepatoma cell), SW480 cell (colorectal adenocarcinoma cell) and the mouse hepatocyte have been observed at the physiology solution environment.2. The shift of the surface of the cells which were exposed to the magnetic field for different time has been observed. We found that the surface damage of the cells exposed to the magnetic field could be observed by AFM before the function of cells has been changed. Compared with other experiment method, AFM observation is a simpler and more sensitive detecting method. Some tiny holes on the surface of tumor cells in the magnetic field were discovered. The sensitivity of suspension cells to the magnetic field is much larger than the adherent cells. Otherwise, compared with tumor cells, the normal cells was less sensitive to the magnetic field. The surface of the K562 cell during different phases of cell cycle was observed.3. AFM results showed that the magnetic field have the damage effects on K562 cell surface.In order to take the research on its mechanism, atomic force microscopy was used to observed DNA in K562 cells which was in different cell cycle phase.4. The damage of K562 cell caused by the magnetic field combined with the anticancer drug was observed. The effects of different anticancer drug on the cell surface were various. For example, the effects of magnetic field combined with taxol or cyclophosphamide on the cells were adduction, but the effects of the cisplatin or the adriamycin combined with magnetic on the cells were synergistic interaction.5. The quality of DNA image was influenced by the concentration of DNA solution. In order to observe the structure of DNA molecular clearly, DNA solution should be diluted. Our suggestion of DNA concentration was 0.03μg/ml. The operator should avoid misoperation that make the DNA strand break.6. The AFM images of DNA extracted from the cells which were radiated with UV have been got. We found that the damage of DNA extracted from K562 cell treated by UVB/UVC presented time/dose dependence. But there were some differences between them:UVB caused DNA strand break in the short time exposure. When the exposure time prolonged, the DNA cross linking appeared. DNA chain appeared with cross linking mainly in the long time of exposure. UVC caused greater damage of DNA than UVB. UVC caused the breaking and cross linking of DNA chain in the short time. When the time of exposure prolonged, the cross linking of DNA was much more apparent. DNA chain was appearance with large cross linking and breaks in the long time of UVC exposure. At the same time it presented some structure destruction such as condensation and enwind.7. The original images of comet structure which observed by AFM can be imaged after wiping off the agarose gel. The structure of DNA of "comet" was observed in situ for the first time:the head of comet was in form of DNA aggregation; the tail of comet was existed in netlike, crossing link and breaks of DNA chains after it was diluted. The results indicated that the guess that "comet" was existed in DNA breaks and loop ring was rationality.8. After the K562 cell were exposed to the magnetic field for 12 hours continuously, the DNA chain extracted from the cells became rude, and appeared a small quantity of DNA cross linking. After the K562 cell was treated by the cis-platin for 12 hours, DNA breaks and the twist structure of DNA have appeared, and existed in beaded form. After the K562 cell was treated by the cis-platin combined with magnetic field for 12 hours, the twist of DNA has appeared, and DNA was damaged heavily. Results of AFM observations indicated that the synergetic reaction between cis-platin and the magnetic field on cells were great.Conclusion:The sample preparation method of atomic force microscope is simple relatively. But there were higher requirements for the kinds and the concentration of fixing agent, and the accurate operating of experimenter was important. Choosing the suitable sorts and the concentration of fixing agent can preserve the fine structure of cells in the living condition. The damage of the cancer cells exposed to magnetic field was aggravated following time. The sensitivity of suspension cells to the magnetic field is much larger than the adherent cells. Otherwise the resistance of normal cells to magnetic field was much larger than the cancer cells. The damage of cells caused by different anticancer drug combined with magnetic field was different. Some were adduction, and some were synergistic reaction. The suitable concentration of DNA solution was required for the AFM observation. The results that got from observing the damage DNA caused by UV indicated that AFM can detect the DNA strand cross linking and breaks directly. The structure of DNA in the "comet" was observed by AFM directly for the first time:the head of comet was in the form of DNA aggregation; the tail of comet was composed of damage DNA that was existed in netlike structure, DNA crossing link or breaks after diluting. The K562 cell treated by the cis-platin combined with magnetic was observed. There were great synergetic effect between cis-platin and magnetic field.