Investigation On Tumor Killing Effect Induced By Dendritic Cells Pulsed With Ar-He Cryoablation On Glioma Cells

Glioma is the most common malignant tumors of the brain, despite the largest surgical resection plus radiotherapy and chemotherapy, relapse is almost inevitable, the prognosis is poor with an average survival of only15-18months. Argon-helium cryo-ablation is a new minimally invasive targeted tumor treatment in recent years, which has been widely used in the treatment of various tumors, such as prostate cancer, kidney cancer, liver cancer, melanoma, breast cancer and glioma, etc. Compared with surgical excision, cryo-therapy not only leads to local destruction of the tumor tissues but also cause anti-tumor immune response, which reduce tumor metastasis and the recurrence rate of the tumor.In the past, the CNS has been described as an immunologically privileged site based on the presence of the blood brain barrier(BBB), lack of conventional lymphatics, low MHC expression and low T cell trafficking. However, with recent advances in understanding CNS immunity it is more accurate to regard the CNS as an immunologically specialized site that can be divided into three different compartments:the brain parenchyma; the ventricles containing choroid plexus and cerebrospinal fluid; and the meninges. The immune reactivity of the ventricles and meninges is similar to that of the periphery. Therefore, the central nervous system can generate an immune response, which provides a theoretical basis for the immune treatment of central nervous system tumors. More and more experimental datas indicate that dendritic cells (DCs) play an important role in the immune response of the central nervous system.Dendritic cells (DCs) are the most powerful antigen-presenting cells (APC) in body and can effectively present antigen to T cells, activate naive T cells, cause anti-tumor effects, which plays the central role of the tumor immune response. Under normal conditions, DCs exist with immature state in the peripheral tissues. After phagocytose the antigen by immature DCs, which process into the antigen peptide and combined with MHC-Ⅰ and MHC-Ⅱ molecules to form antigen peptide-MHC molecule complex and expressed on the surface of DC, while increase expression of the DC costimulatory molecules (CD80, CD86) and adhesion molecules to form mature DC. Mature DC can identify and activate tumor antigen-specific T cells to proliferate, which secrete a large amount of cytokines such as IL-12, leading to tumor degression.Whether necrotic tumor tissue formed by local cryo-therapy can regard as an effective tumor antigen, which be uptaken by DC, contributing to the activation of T lymphocytes is critical problem of tumor-specific immune response. Studies have shown that the cryo-therapy treatment is an effective means to produced tissue necrosis. Necrosis and damage include the damage of cell membrane and the release of internal cytoplasmic organelles and membrane. This process is a strong signal for cellular immune stimulation. Meanwhile necrosis tissues lead to more cytokine release. So the activated possibility of antigen presenting cells is greatly increased. Unlike thermotherapy, cryotherapy does not produce a lot of damage for inside tumor antigen peptide. These relatively complete tumor-specific antigen peptide are released into the surrounding environment, it is easily uptaken by antigen presenting cells and presented to T cells to generate immune response. In recent years, a large number of experimental studies in vivo showed that freezing and thawing of frozen tumor tissue products can promote DC maturation and antigen presenting function, and can active T lymphocytes to produce effective anti-tumor immune response.Currently, tumor antigen pulsed DC as vaccine has been widely used in clinical and achieved good results. The types of tumor antigen and loading methods of DC vaccine include tumor antigen peptides sensitized DC, tumor cell protein extracts stimulated DC, DC fused with tumor cells, tumor-derived cell DNA/mRNA sensitized DC, cytokines and chemokines genetically modified DC and so on. Ideal tumor antigens of DC vaccine should be capable of inducing a polyclonal response against different epitopes of tumor antigens, do not induce an autoimmune response, no safety problems, the patient is not restricted with MHC types, ease of preparation and application. The use of tumor cell lysates pulsed DC, can avoided shortcomings of a single antigen which may not completely kill the tumor cells, so the use of freeze-thaw lysis of tumor cells pulsed DC has good application prospects for glioma treatment.Therefore, based on the powerful ability of antigen-presenting by dendritic cells, the mouse GL261glioma cell lines were selected for this study. First of all, we use argon-helium thaw-frozen glioma cells, extracted tumor antigens, plused with C57BL/6mouse bone marrow-derived DC. Expression of DC surface molecules was detected by flow cytometry, to prove whether freezing products can promote DC maturation, detect changes of cytokine secretion released by DC. T cells extracted from mouse bone marrow and were cultured with DCs to detect the activity of T cells, simultaneous detect IFN-y levels secrete by T cells to evaluate DC antigen presenting function. The activated T cells were co-cultured with GL261glioma cells, detection of T cell killing rate of GL261glioma cells by different effector and target ratios, observe killing effect of T cells to tumor cells.Chapter Ⅰ Isolation and Culture of C57BL/6mice’s dendritic cellsObjective:To establish a method of isolating and culturing dendritic cells (DCs) from the bone marrow of C57BL/6mouse.Methods:Dendritic cells were obtained from the bone marrow of C57BL/6mouse’s limbs by RPMI-1640lavation were cultured in the RPMI1640medium in vitro under the condition of20ng/ml granulocyte-macrophage colony-stimulating factor (GM-CSF) and20ng/ml interleukin-4(IL-4). Tumor necrosis factor-alpha (TNF-α,100ng/ml) was added into the medium in day5to stimulate its maturation. The morphological changes of DCs were observed under inverted microscope. Flow cytometry was used to detect the expression of surface molecules (CD80, CD86, MHC-Ⅰ and MHC-Ⅱ) on DCs on day5and day8.Results:DCs could be isolated from C57BL/6mouse bone marrow by GM-CSF and IL-4. Typical DC morphologies can be observed in cultured day eight. DC expressed surface antigens, including CD8011.98%, CD864.08%, MHC-Ⅰ6.90%, MHC-Ⅱ37.68%on the7th day; CD8097.40%, CD8691.99%, MHC-Ⅰ97.25%, MHC-Ⅱ98.71%on the10th day.Conclusion:The culture method of DC from bone marrow of C57BL/6mouse has been successfully established, which provide sufficient cell source for DC immunotherapy against glioma. Chapter II Investigation on Tumor Killing Effect Induced by Dendritic Cells Pulsed With Ar-He Cryoablation on Glioma CellsObjective:To explore the effect of Ar-He cryoablation on glioma cells to maturation of bone marrow-derived dendritic cells (BMDCs) in C57BL/6mouse, and the mechanism of dendritic cells (DCs) induce tumor killing effect.Methods:Ar-He cryoablation on GL261glioma cells for experimental group, only insert probe to GL261glioma cells for negative control group, Ar-He cryoablation on the same amount of PBS for blank control group. The protein concentration of freezing and thawing products was measured. Dendritic cells were induced from mouse bone marrow in vitro. After tumor cell lysate was added, morphological characteristic of DC was observed under inverted microscope, the expression of surface markers CD80, CD86, MHC-I and MHC-II was analysized by flow cytometry. The concentration of IL-6, IL-1β, tumor necrosis factor (TNF)-a, IL-12in the supernatants were detected by ELISA. T cells were extracted and co-cultured with DC, the proliferative activity of T cells were detected using CCK-8. The amount of IFN-y secreted by DC was detected by ELISA. The killing rate of T cells to GL261glioma cells was detected using CCK-8at different effect-target ratios.Results:The protein concentration of the experimental group was higher than control group (P<0.01);The experimental group showed the typical characters of mature DC, the expression of DC phenotype CD80,CD86, MHC-I and MHC-II was higher than control group (P＜0.01). The concentration of IL-6, IL-1β, tumor necrosis factor (TNF)-a, IL-12secreted by experimental group DC were higher than control group (P＜0.01); The proliferative activity of T cells in the experimental group was higher in the ratio of1:10and1:20compared with the negative control group and blank control group, the difference was statistically significant (P＜0.01); The amount of IFN-y secreted by DC in the experimental group was higher compared with the negative control group and blank control group, the difference was statistically significant (P＜0.01). The killing rate of CTLs to GL261glioma cells in experimental group was significantly higher than control group at different effector-target ratios (P<0.01).Conclusions:A large amount of tumor antigen can obtained from Ar-He cryoablation on GL261glioma cells which can induce maturation of DC. These DCs can produce anti-tumor effect after pulsed with tumor lysate, which provides an experimental and theoretical basis for glioma argon-helium cryogenic target immunotherapy.