| Background:Chronic myelogenous leukemia disease is a common malignant proliferate cells,original from hematopoietic stem cell.The therapy result of leukemia is not good.One major reason is the cell's drug resistance to chemotherapeutic drugs.At present,The key research direction of leukemia treatment is to search for a new treatment technology.It has been known that Micro/nano-medium magneticfield hyperthermia technology can be used to the treatment of blood tumor.Moreover,it can obviously inhibit the growth of human leukemia cells in vitro,but the specific mechanism of inducing apoptosis is not very clear.It has been known that cell-penetrating peptides can get into cells carrying Fe3O4 nanoparticles.Mitochondria and nuclei are two important organelles in the cell.So,We suggest that the enrichment of magnetic nanoparticles in mitochondria and nuclei is beneficial to the effect of magnetic induction hyperthermia.Wedesigned the mitochondrial and nuclear targeting peptide sequences and combining the CPPs transmembrane sequence with them,guiding the magnetic nanoparticles to the designated site in the cell.Objective:1.To explore the possibility of magnetic hyperthermia inducing apoptosis of chronic granuLocytic leukemia resistanting to Gleevec K562/G cells and to explore its mechanism of action.2_The preparation of signal peptide coupled magnetic nanoparticles and to investigate the safety and efficacy of the nanoparticles used in magnetic hyperthermia at the celluLar level.Methods:1.We used the chronic granulocytic leukemia resistanting to Gleevec cells K562/G as the experimental materials,the stainless steel hollow balls were used as the medium.The cells were heating in 43? for 30min under the magnetic field of 300kHz 40Gs.We detection the rate of cells apoptosis,mitochondrial membrane's potential and the blocking condition of cell cycles.Toexplore whether the cell's death were caused by apoptosis;We conducted magnetic hyperthermia on cells in 43?,47?,50? for 30min,and tested caspase3,8,9'sactivity by colorimetric assays.Then we analysed the expression of Hsp70 and ?-catenin use Western blotting,Immunofluorescence were used to comparing thechanges in the ratio of Bax/Bcl-2.2.We got 4 kinds of targeting peptides RM,RN,TM and Protamine.The Fe3O4 magnetic nanoparticles were synthesized by chemical co-precipitation method.We coupling the four kinds of targeting peptides to the surface of synthesized Fe304 magnetic nanoparticles.The physical characterizations were carried outby TEM,FTIR and Zeta potential analyzes,and drawed the VSM curve.Cells incubated with Fe3O4 targeting nanoparticles in four concentrations of 0,8,16,24mg/mL.After24h,we detected the cell's compatibility of nanoparticles in MTT method.The iron content in one cell were detectedby ICP.Confocal microscopy was used to determine the targeting of modified particles.Finally,the MTT method was used to detect the ability of modified particles to inhibiting the proliferation of the cells in the same temperature.Results:1.The temperature of stainless steel hollow spheres will rise under the magnetic field of 300kHz,40Gs,in the concentrate of 35?30?25?20?15mg/mL.The results of MTT showed that hollow spheres have less cytotoxicity and good cytocompatibility after 24h,48h,72h's incubation with cells.After 30min's heating,the cell's appreciation rate of 43?was73.46±2.60%;47? was 66.77±2.34%;50? was 55.66±3.24%.The technology of magnetic hyperthermia can efficiencyly kill K562/G cells and inhibit its proliferation.After 30min's heating,under the AMF in 43?,We detected the apoptosis rate of K562/G by Flow cytometer and it reached 39%.The proportion of cells that took part in the mitochondrial membrane potential depolarization increased from 10.2%to 70.8%.The cell cycle was blocked in the phase of G2/M.Theactivity of Caspase3,8,9 was significantly enhanced after magnetic induction hyperthermia in the 24h,and with the increase of heating temperature,more Caspase3,8,9 zymogen was activated.West blotting showes that the expression of HSP70 increased with the increase of hyperthermia temperature,and the expression of ?-catenin decreased.The ratio of Bax/Bcl-2 was increased with the increase of hyperthermia temperature.2.The diameter of Fe3O4 magnetic nanoparticles were about 10nm or so,after modified with signal peptides the diameter size and shape are notly changed.The resuLts of MTT showed that modified nanoparticles have less cytotoxicity and good cytocompatibility.The zeta potential increased,magnetic saturation decreased.The resuLt of FTIR showed that the modified nanoparticles have thecharacteristic absorption peaks of Fe-O-Fe,Si-O,-CO-NH-,the signal peptides were successfuLly coupled to the surface of Fe304 magnetic nanoparticles.TheresuLts of ICP showed that after coupling signal peptides to the nanoparticles the content of Fe in single cell increased significantly.We can see by Confocal laser scanning that Fe3O4 magneticnanoparticles can pass through the cell membranes under the guides of RM,TM and gathered in the mitochondrial.When we hatched SW480 cells with Fe3O4 magnetic nanoparticles in an alternatingmagnetic field of 43? for 30min,the inhibitory effects of modified nanoparticleshasbeen significantly enhanced,the best of them is PRO-MNPs,cell survival ratewas only 29.6%.Conclusions:Preliminary explanation that magnetic hyperthermia can activate the mitochondrial signal pathway,leading to apoptosis of K562/G cells.It provides a goodfoundation to further exploring the anti-tumor mechanism of magnetic hyperthermiatechnolog.At the same time,it is found that the magnetic nanoparticles modifiedby signal peptide are safe and reliable,It significantly improved the efficiency of hyperthermia when applied to magnetic hyperthermia. |
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