| Objective:The aim of this study is to investigate the implications of dendritic cells-mediated vaccine therapy for intracranial gliomas in rats.1. To establish stable and reliable glioma-model of rats and to know about the immunological state of hosts;2. To prepare and select highly effective dendritic cells-mediated vaccine;3. To treat gliomas of rats in vivo with dendritic cells-mediated vaccine.Methods:1. To establish a C6 brain-glioma model in rats: Rat C6 glioma cells were cultured in vitro and then the C6 cells at logarithmic growth phase were harvested, washed 3 times in PBS, subsequently, resuspended in serum-free RPMI-1640 medium containing 10g/L agarose. C6 tumor cells ( 5×105 ) were then injected intracranially under the stereotactic monitor into the right caudate nucleus of the Wistar rat brain. Implantation was followed by general observation and MRI scan; Furthermore, the rats with implantation were sacrificed at different experimental day, trans-aorta paraformaldehyde perfusion was conducted at 7, 14, 21, 28 and 35 days following the implantation and before the natural death. Brains and tumor were sectioned and inspected. The tumor containing samples were prepared histologically for hematoxylin and eosin ( HE ) staining and immunohistochemical staining about GFAP. In addition, we detected the level of CD8+T lymphcytes from peripheral blood of rats harboring intracranial glioma.2. To prepare and select highly effective dendritic cells-mediated vaccine: The precursors of dendritic cells were isolated from bone marrow of rats, stimulated in vitro with recombinent rat granulocyte-macrophage colony-stimulating factor (rrGM-CSF) and interleukin-4 (rrIL-4). Then rat DCs were pulsed with C6 tumor cell antigens prepared with different methods: freeze-thawed tumor cell lysates, total protein extracted from ultrasonic crushed tumor cell or heating-induced apoptotic tumor cells. Subsequently primed DCs were cocultured with T lymphocytes from spleen to induce CTL. Lymphocyte chemoattractant factor from DCs and cytokine IFN-γ release were determined by ELISA, the cytotoxicity of CTL was assayed by JAM test.3. To treat rats harboring intracranial C6 tumor with dendritic cells-mediated vaccine: DCs pulsed with apoptotic C6 tumor cells in vitro were injected subcutaneously into rats harboring intracranial C6 tumor. Rats from different group were treated with five weekly subcutaneous injections of either control media, unpulsed DCs, or DCs pulsed with apoptotic tumor cells. The animals were followed for survival, volume of tumor by MRI. CD8+ T cells, cytotoxicity assay in vitro and proliferational function in peripheral blood were determined by FACS.Results:1. Our results indicated that intracranial growth occurred in all implanted rats ( 100% ); Furthermore immunohistochemical analyses were used to document an expression of GFAP on the surface of implanted tumor cells which convinced that the implanted tumor derived from glias. The development of tumor speeded up evidently from the third week after implantation. The level of CD8+T cell lowered significantly as the time of harboring tumor prolonged (21.93±3.81%), P=0.004 as compared with normal rats (27.92±1.30%).2. Studies in vitro showed that we can obtain DCs hving "dendritic processes" of typical morphological characteristics using our cultural system. The surface of DCs expressed high level of CD11c, OX6, OX62, MHCII molecules and relative low level of CD80,CD86 costimulatory molecules which were the characteristics of immature DCs and suitable to prepare the tumor vaccine. We found that in the three kinds of tumor vaccines the DCs pulsed with apoptotic tumor cell in vitro could induce an enhanced ability of T-cells proliferation and cytotoxic T lymphocyte activity. In addition, significantly enhanced concentration of IFN-γand chemoattractant factor compared with other groups were observed in rats treated with apoptotic glioma cells pulsed-DCs.3. We observed that C6 glioma model rats treated with apoptotic tum...
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