Experimental And Mechanism Study On Immunotherapy Of Human Brain Mali-gnant Glioma With Dendritic Cell Vaccine Targeting Of Cancer Stem Cell In Vitro

OBJECTIVE:At present,DC-inducing anti-tumors immune has become one of important cancer immunotherapy.The DC vaccine loaded by hyperthermia-induing apoptotic glioma cells antigens can enhance differentiation and maturation of the dendritic cells, strengthen its function of antigen presentation and induce stronger tumor killing effect of cytoxicity T lymphocytes. To explore the method of culturing human glioma stem cell in U251 cell line in vitro with reformed neural stem cell culturing medium, meanwhile to study biological characters on proliferation and differentiation, and so on. To explore anti-glioma cells ability and mechanism of specific T cells induced by dendritic cells loaded with antigen of glioma stem cells,in vitro.METHODS:The murine bone marrow-derived DCs were induced and amplified by rmGM-CSF and rhIL-4 using improved Inaba way in vitro, the biological characteristics of which were indentificated by electron microscope and flow cytometer. Human U251 glioma cells were cutured in vitro and induced by hyperthermia 43℃,44℃,45℃for 1,2,3,4 hours respectively and then routine culture for 12 hours.The DCs were sensitized by apoptotic U251 cells to prepare DC vaccine in advance which was charactirizated through the mixed lymphocyte responses and cell killing experiment. Human glioma cells U251 were cultured in serum free mediam DMEM/F12, plus LIF(10ng/ml), EGF(20ng/ml), FGF(20ng/ml) and B27(20ul/ml), examined daily and subcultured weekly. Cell spheres were collected after 4 weeks.The expressions of neural stem cell marker Nestin and CD133 were detected by immunocytochemistry. CD133+ cells and nestin+ cells were detected with flow cytometry, in adherent U251 and non-adherent cell spheres, which were cultured for 1 to 8 weeks. After 4 weeks, Cell spheres were collected at 4 weeks and incubated for 5 to 10 days for differentiation in medium containing serum. Then, the expressions of NSE, MAP2 and GFAP on differentiation cells were examined by immunocytochemistry. Cancer stem cells were cultured from clinical specimen of glioma,lysate of glioma stem cells was obtained by the repeated freezing and thawing method,and DC stemed from mesenchymal stem cells(MSCs). Glioma stem cell vaccine was got by mixing tumor lysate with DC,then,surface molecules of DC were checked by flowcytometry. CCK-8 method was used to delect the DC vaccine's ability of promoting T cells proliferation and killing glioma cells. Level of IFN-yin the supernatant was checked by ELISA.RESULTS:The DCs were generated after 6-7 days induce with rmGM-CSF and rhIL-4, the typical dendritic structure on membrane surface was indentificated by inverted microscope and electron microscope.The flow cytometry confirmed that DCs expressed the typical surface marker CD11c and functional correlation antigen CD80,CD86 and MHC-II in high level meanwhile. The best condition of glioma cell inducing apoptosis was 44℃for 3 hours and then routine culture for 12 hours,which was defined by inverted microscope and flow cytometry. Apoptotic tumor cells could preferably load DCs.The DCs vaccine had strong ablity to stimulate the homogeneity mixed lymphocyte responses.Apoptotic tumor cells loaded DCs also could stongly active antigen specific CTL to kill glioma cells. A small amount of U251 cells had capacity of continuous proliferating and renewing for long time, in vitro, with positive immunohistochemistry-staining on Nestin and CD 133. The percentage of CD 133+ cells were 0.1% in adherent U251,1.5% after 1 week,3.0% after 2 weeks and 10.0% after 8 weeks in non-adherent cell spheres. Glioma stem cells could differentiate into neurons and astrocytes. Differentiation cells were positive for neural marker MAP2/NSE or glial marker GFAP, some expressed both markers. After stimulation of lysate of glioma stem cell, expression of surface molecules of DC was up-regulated,including CD80,CD86,CD11c and MHC II. Glioma stem cell vaccine could stimulate T cell proliferation strongly. The vaccine could induce effective immune response, kill tumor cells and boost the secretion of interferon-y.CONCLUSIONS:The functional DCs were successfully indced and amplificated by cytokines rmGM-CSF and rmIL-4 in vitro.The DCs vaccine loaded by hyperthermia-inducing apoptotic tumor cells could stimulate autologous T lymphocyte proliferation, and induce antigen specific CTL produce strong killer function. Human glioma stem cells could be cultured from U251,and could be differentiated into neurons and astrocytes, as neural stem cells,which played an important pole in the pathogenesis of glioma. Dendritic cell vaccine can effectively induce specific cytotoxic T cells, and show strong anti-tumor properties, loaded with antigen of glioma stem cells,in vitro.