A Study Of Making DC Tumor Vacine By High Intensity Focused Ultrasound And Observing Its Antitumor And Promoting Tumor Cells Apoptosis Effect

Objective①To 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 feasibility of high intensity focused ultrasound in making tumour antigen and DCs vaccine. And investigate the active antitumor immunoprotecitive effect after inoculating mice by this production.②Furthermore to break a new way of anti-tumor immunoprophlaxiy and immunologic therapy mediated by DC.③To investigate the apoptosis of Tumor cell, and explore its therapeutic mechanism, To open up new areas of application for HIFU treatment on tumors.Methods①With IL-4 and GM-CSF, the cells from mouse bone marrow were④electron microscope on 3-7 days after culture.;Flow cytometry was used to detecting the expression of surface molecule (CD86 CD80 and I-Ad H-2kd) in DC.②fter irradiate cultured Ct-26 tumor cells by different doses of HIFU,the number of survival cells was detected with MTT colorimetry. To observe the morphology change of the cell with typan blue staining., and concurrently therefore chase down the HIFU dose to peoduce tumor antigen.③The cultured CT-26 tumor cells were irradiated by dose of 1000W/cm2×30s,then acquired tumor antigen,and this antigen was co-incubated with DC that had been cultivated for 5days in vitro so as to make DC tumor vaccine.④The active immunoprotecitive effect of DC tumor vaccine in vivo:to establish four groups:the HIFU group (group A), the repeated freezed group (group B); the simple DC group (group C), and negative control group (group D). normal BALB/c mice were inoculated CT-26 colon cancer cells(5×105DC cells one mouse)subcutaneously, after seven days, the mice were vaccinated by the acquired DC tumor vaccine(5×105DC cells one mouse)one-time, seven days later, the mice were vaccinated by the acquired DC tumor vaccine(5×105DC cells one mouse)one-time again. After 7 days execute the partial mice to take out the tumor to survey tumor weight, the mouse dispel the lump net weight and the tumor volume, the surplus mouse uses to observe the medium survival time, the survival rate and so on..To reach whether there is differences in groups.⑤The research of DC vaccines promote tumor cell apoptosis:mouse tumors were stripped so as to make single cell suspension and tissues simplce. AnnexinV-FITC/P respectively and TUNEL method were used to reach tumor cell apoptosis and compare the differences in groups.Result①After 3 days cultivation with IL-4 combined GM-CSF, mouse bone marrow cells showed remarkable modality changes, include morphological abnormity, cluster formation. After 6-8 days, there have been shown burr-like prominency and elongate that which present the typical character of DC. Flow cytometry detected the high expression of CD80,CD86,H-2Kd and I-Ad.②With the different doses of duration fixed HIFU irradiation(30s), when intensity elevated, the survival ratio of CT-26 cells descended. There have been shown a exponent regress relationship; With the different intensity of duration fixed HIFU irradiation (1000W/cm2); when duration elevated, the survival ratio of CT-26 cells descended. There have been shown a consistent linear correlation. The HIFU dose of 1000W/cm2×30s resulted in complete rupture of cells and total cell death. Therefore, the dose of 1000W/cm2x30s was considered the appropriate for producing tumor cell rupture solution (tumor antigen).③Compared HIFU group and freezing group with negative group, there have been shown remarkable difference intumor weight and size at 20 days after seed (p<0.01 or p<0.05); Compared HIFU group with freezing group, there have been remarkable difference in tumor weight and size too. Mice medium survival time, survival rate of HIFU group, To compare survival rates among three groups, difference was remarkable.These indicated that DC tumor vaccine derived by HIFU and repeat freezing may effectively inhibit the proliferation. Notable difference was also observed between HIFU group and freezing group, more effective effect was seen in HIFU group.④The HIFU group, Therepeated freezing group with the simple DC group and negative control group of tumo irradiated group and the repeated freezing group of tumor cell apoptosis differences were also statistically significant (P<0.05). The DC vaccines prepared by the HIFU and repeated freezed can resisttumor growth and promote the tumors apoptosis, thereby role in the treatment of tumors, but the former is better the latter.Conclusion①Cultivation of mouse bone marrow cells by GM-CSF associated with IL-4 could enlarge mature DC according with its character.②HIFU irradiation resulted in inactivation and rupture of tumor cells. Tumor antigen produced y HIFU could sensitize DC in vitro, and then made DC tumor vaccine.③HIFU applied a better approach of active immunity in mice against tumor occurrence.④Tumor vaccine prepared by HIFU have a certain therapeutic effect on mice tumors and can promote tumor cell apoptosis, and better than tumor vaccines prepared by the repeated freezed.