A Stduy Of High Intensity Focuse Ultrasound In Loading Tumour Antigen Of Dendritic Cells And Lead T Cell To Secrete Interferon-gamma In Vitro

Objective To establish a method of expanding dendritic cells from mice bone marrow in vitro and to identify it with morphological, immunological phenotype determination. To investigate the possibility of high intensity focused ultrasound in making tumour antigen, loading DC and making DC vaccine. This research will open up the new application for HIFU.Methods①With IL-4 and GM-CSF, the cells from mouse bone marrow were cultured. The morphological changes were observed under light microscope and electron microscope on 3- 7 days after culture. The expressions of CD83 and CD86,H-2Kd and I-Ad were test by flow-cytometric analysis.②Regular irradiation time, after irradiating cell line CT26 with sound intensity of HIFU, the number of survival cells was detected with typan blue staining and MTT colorimetry. Analyse the relation of HIFU dosage and tumour cell's survival rates. To observe the morphology change of the cell with typan blue staining and find out the relationship about dosage-effect.③Regular irradiation time and the power of ultrasound,the ultrasound doses leading to acute cell death were figured out according to the dose-effect relation curve. After culturing 48h T and DC pulsed with tumour antigen made by HIFU, we detected cytokines IL-12 IFN-γwith ELISA, compare with freezing melting,CT26 and control.Result①With IL-4 and GM-CSF, morphological changes were found and dendritic protrusion appeared on 3rd day after culture; dendritic protrusion prolonged on 5th day after culture; flow-cytometric analysis showed that expressions of CD83 and CD86,H-2Kd and I-Ad.②With increasing dose of sound intensity,cell survival rate decreased sharply and more cell debris was observed. It was found that 1000W/cm2×30s was the best dose bringing complete cell destrution. There is not cell that survives, the tumour cell totally loses cell's shape,all broken into chip.③Higher levels of IL-12,IFN-γwere detected in HIFU freezing melting and CT26 groups, there was significant differences (p<0.05) in the three groups and control. The levels of IL-12. IFN-γin HIFU,freezing melting groups is higher than CT26 group, there was significant differences (p<0.05). But it was no significant differences compare HIFU and freezing melting group(p>0.05).Conclusion We could isolate and culture dendritic cells from bone marrow of Balb/c mice. Dendritic cells of mice have typical morphology and characteristic phenotype. The results suggest that high intensity focused ultrasound was capable of destroying and fracturing living tumor cells at one time. Tumour HIFU antigen could load and mature DC. Mature DC secreted a large of IL-12 and could stimulate T cell to secrete a large of IFN-γ.