| Gliomas are the most common malignant brain tumors, average44.69%of all malignant central nervous system tumors. Because glioma is diffusely invasive, highly infiltrative tumors spreading into surrounding normal brain tissue, it is not feasible for complete surgical resection of gliomas. Despite advances in surgical techniques and adjuvant chemocherapies or radiation, the prognosis for patients with gliomas remains poor, with a median survival of14months.Argon-helium cryoablation technology rises from the United States in recent years. The argon-helium cryosurgical system which relies on the principle of the Joule-Thomson effects to freeze tumors. There is a cryoprobe with a diameter at a temperature of-140℃in a single freeze-thaw cycle. The temperature of frozen tumors reach-140℃very quickly resulting in an ice-ball. The area exposed to freezing will show necrosis. It shows the immediate formation of an ice-ball in diameter around the cryoprobe. Cryosurgery is a minimally invasive surgery. Its efficacy and safety require more observation by experimental and clinical practice. With the recent advance out worldwide, this technique allows precise cryoablation of a wide variety of liver cancer, kidney cancer, pancreatic cancer, prostate cancer, lung cancer, and breast cancer. Due to its technical ease and minimal morbidity, the use of cryosurgery to ablate tumors is expanding. A potential secondary advantage to the freezing of malignant disease is the cryo-immunologic response, the generation of an anti-tumor immune response triggered by the natural absorption of the malignant tissue. While initially proposed based on clinical observations of distant disease regressing after cryoablation of a primary tumor, results from preclinical studies have been mixed and the existence of a cryo-immunologic response has been controversial. Potential mechanism by which cryoablation may modulate the immune system were unclear. Recent studies show that the mechanism is concerned with Antigen presenting cells(APC), which strong ingest and process antigen.Dendtritic cells (DCs) are professional antigen presenting cells in vivo. It has been shown that dendritic cells can efficiently phagocytosis various forms of tumor cells,including live, apoptotic, necrotic tumor cells and acquire tumor antigens from the phagocytosis tumor cells. DC processe exogenous antigens typicallyd into peptides, load major histocompatibility complex class Ⅱ (MHC Class Ⅱ) molecules and transport the cell surface for recognition by naive antigen-specific T-cells. Activation of these T-cells dependent upon not only recognition of the antigen by the T-cell receptor (TCR) but also co-stimulatory signals, such as CD28on the T-cell recognizing B7.1(CD80) or B7.2(CD86) on the DC, uniquely capable of inducing both primary and recall immune responses,and they are key to stimulating clinically meaningful antitumor immunity.Recent clinical studies have shown that immunotherapeutics with DC-based cancer vaccines have the potential to improve patient outcome for a wide range of tumor types.It has been reported that DC vaccination loaded with tumor-associated antigens could induce tumor-specific CTL against multiple epitopes. Increasing evidence has demonstrated that DC,when loaded with tumor antigens,are able to process the loaded antigens and present antigenic epitopes to T cells,resulting in the induction of tumor-specific CTL and antitumor immunity.To date,many methods have been developed or evaluated for loading tumor antigens to DC. There are several antigens to load DCs, such as apoptotic and necrotic tumor cells, cell lysates, purified protein, peptide, plasmid cDNA et al..Glioma is an organ composed of a heterogeneous combination of tumor cell types, and every tumor cell in a glioma are different in their ability for proliferation, differentiation and sensitivity to anti-tumor drugs. The most appropriate host anti-tumor T cell response requires a series of epitopes, rather than confined to a particular epitope, therefore, it is a simple and effective method to prepare DC vaccination by loading DCs with the whole tumor cells.Up to now, DC vaccination loaded with cryoablative tumor-associated antigens has been successfully applied in many tumors. Because of the presence of glioma heterogeneity, it is very difficult to get specific glioma antigens. It has been demonstrated that vaccination with DCs pulsed with tumor lysates obtained by rapid freezing and thawing of Ar-He Cryoablation is more effective than other antigen-pulsed DCs.Compared with traditional vaccines, cryoablative tumor-associated antigens vaccine can induce the body’s whole immune responses, and it can increase vaccine targets. Second, the identity of the tumor antigens and the human leukocyte antigen (HLA) of patient does not need to be known and the presence of multiple tumor antigens reduces the risk of antigen-negative escape mutants; Therefore, DCs transfected with cryoablative tumor-associated antigens is a simple, feasible and safe method for tumor immunotherapy. For a long time, as the blood-brain barried (BBB) had been thought to prevent components of the immune system from entering, the central nervous system (CNS) has traditionally been regarded as an immunologically privileged site. However, many researches have demonstrated, under pathological conditions, that lymphocytes could cross blood brain barried (BBB) and induce immunological reactions in the CNS. Therefore, immune responses could be involved in the central nervous system diseases, which provide a theoretical basis for anti-tumor immunotherapy in the CNS.In this study, we performed argon-helium cryosurgeries in C6gliomas.Then, total cryoablative tumor-associated antigens from C6cells were extracted, and DC vaccination with total cryoablative tumor-associated antigens cells were prepared; Finally, we immunized C6cells/Wistar rats by DC vaccination, the effects of DC transfected with cryoablative tumor-associated antigens were observed.we aimed to explore the mechanism of angon-helium cryosurgery in relation to the cellular immunity and provide experimental evidences for the clinical application of this technique in glioma treatment.The study includes two chapters:Objective:To establish a simplified culture system from rat C6cell line. Using Ar-he Cryoablation System performs the procedures of cryoablation of rat glioma.Method:Rat C6gliomasarcoma cells were cultured in serum-containing medium (DMEM/F12+10%FBS). Choose a rat C6glioma lines, application including serum culture medium (DMEM/F12+10%FBS) training. Preparation rats C6brain tumors cells suspension liquid, conventional two-cycle argon helium refrigeration method for rats glioma whole cell freeze-thaw antigen and-20℃cryopreservation.Result:Rat C6gliomasarcoma cells were cultured in serum-containing medium as adherent cells, they grow processes just like fibroblast, showing short spindle, long spindle-shape, star-shape and long fiber-shape. Opening argon helium refrigeration for C6gliomasarcoma cells results showed the immediate formation of an ice-ball in diameter around the cryoprobe.Conclusion:Argon helium refrigeration prepareted rats glioma whole cell freeze-thaw antigen, and the top of probe form a range of ice hockey, which is formed within the tumor cells in ice crystals. Ice crystals in hot and cold stimulation burst of cell membranes and the entire tumor cells to produce direct mechanical damage, make tumor cells apart necrosis.Objective:To establish a method of isolating and culturing dendritic cells (DCs) from the bone marrow of Wistar rats.To investigate whether bone marrow-derived dendritic cells (BM-DCs) pulsed with tumor lysates (TP-DCs) obtained by rapid freezing and thawing of Ar-He Cryoablation mediate immunotherapy against intracranial gliomas in rats.Method:Bone marrow-derived mononuclear cells (MNCs) were first prepared from tibia and femur bones of wistar rats. These cells were cultured with cytokines GM-CSF, IL-4, and TNFa to induce their maturation, which were purified with attachment method. BM-DCs were pulsed with or without tumor cell lysate obtained by rapid freezing and thawing of Ar-He Cryoablation at a1:3DC:tumor cell ratio. Finally, they were analysed by morphological observation and phenotype analysis. The IL-12level in the supernatant of DCs was detected by ELISA. In order to determine whether or not vaccination with C6TP DCs induce the therapeutic potential in the established glioma-bearing model, the C6cells cultured in vitro were stereotaxically implanted into the left caudate nucleus of Wistar rat brain. glioma-bearing rats were injected with vaccination with DCs on days3and10. Survival time was observed and determined using the method of Kaplan-Meier analysis.Result:DCs from rats’bone marrow cells cultured with cytokines and pulsed with tumor lysates for9days showed the character of typical mature DCs. Morphologically, these cells were large with oval or irregularly shaped nuclei and with many small dendrites. Phenotypically, FACS analysis showed that they expressed higher levels of CD80, and CD86antigen(71.81±1.10%、74.66±1.48%in TP-DCs group vs.49.49±1.08%、51.20±2.06%and48.47±1.09%、49.53%±1.89%in DCs cultured with gliomas groups or PBS, P<0.05). Functionally, the IL-12level in the supernatant of DCs had shown to increase obviously(245.99±3.20pg/ml in TP-DCs group vs.138.68±3.20pg/ml and135.16±2.88pg/ml pg/ml in DCs cultured with gliomas groups or PBS, P<0.05). These cells gained the capacity to mediate immunotherapy against intracranial gliomas in rats (Median survival dates of33days in TP-DCs group vs.22,24days in DCs cultured with PBS or gliomas groups, P<0.05). However, there was no significant difference between the groups of DCs cultured with PBS or gliomas in the expressed levels of CD80and CD86antigen, produced levels of IL-12, and the ability to induce effective immunity against gliomas. ConclusionConclusion:The culture method of DCs from bone marrow of Wistar rats has been successfully established, which provide cell source for DC immunotherapy against glioma. BM-DCs pulsed with tumor lysates obtained by rapid freezing and thawing of Ar-He Cryoablation, as a new therapeutic Vaccine, can mediate immunotherapy against intracranial gliomas in rats. |
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