| Ovarian cancer, one of the three malignant tumors in women, has the worst prognosis of all gynecological cancers and is a leading cause of death from cancer in women. Nowadays the routine treatments on ovarian cancer include surgery, chemotherapy and radiotherapy. Because of their limitations, more effective and specific ways in killing tumors are needed. Biological treatment has received a huge amount of attention in recent years. As a type of treatment that works with immune system,Biological treatments involving non-specific cytokines (IL-2, IFN-a) and target therapy of monoclonal antibodies and ap cytotoxic T cells (CTL) help to eliminate tiny tumors and remnant pathological changes. In contrast to non-specific cytokines, the great virtue of target therapy that monoclonal antibodies and ap T cells precisely recognize the tumor antigen restricts the application of monoclonal antibodies andαβT cells in clinical treatment. The acquirement of y8T cells ex vivo is a mature technology; however, there are still some problems. For example, the numbers and immunological activities of seed cells in the patients with median or late-stage malignant tumors or treated with chemotherapy decreased and are not up to quantities demand for treatment. Therefore, gene-modified y8 T lymphocytes consider being prepared for tumor therapy using genetic engineering methods.In recent years, our lab selected the CDR3 of 82 chain (CDR38) as a research target and investigated the interaction of CDR3 peptide or CDR3 grafted Ig with tumor cells, tumor tissues or tumor cell protein extracts. Our results suggested that CDR3δwas the important position for antigen recognition and furthermoreγδT cells were able to widely recognize and kill many solid tumors through CDR3 of 8 chain.In the first part of my thesis, we constructed gene modified y8 T cells whose CDR38 is replaced by sequences of OT3 and showed they had tumor specific reactivity and cytotoxity in vivo and in vitro. Our data indicated that the adoptive transfer of gene modified y8 T cells can provide a new strategy for cancer therapy.The first part of the experiments contained the following work1 Create technological platform for screen the tumor specific and reactive CDR3 region.Linearized plasmid pGEM4Z/EGFP/A64 was template for in vitro mRNA. Compared the electroparation expression rates and cell survival rates under various parameters, we make sure the prior condition and build a platform of mRNA elctroparation. Stimulated PBMC were transferred by mRNA electroporation with the combination of the. full length ofδchain with different CDR3 region and the full length ofδchain from ovarian cancer infiltrated yδT cells and expressedγδTCR whose CDR3 region of 8 chain were replaced by OT1, OT3 or OT10 sequences, named y9δ2(OT1)T cells,γ9δ2(OT3)T cells and y9δ2(OT10)T cells separately. After stimulated by various tumor protein extracts,the transfectant cells expressing y8 TCR with specific CDR3 sequences secreted TNF-a, IFN-y. At the same time, transfentent cells showed the specific lysis against tumor cell lines. y9δ2(OT1)T cells are suboptimal for the cytotoxity and cytokines secretion in comparison to y9δ2(OT3)T cells and y982(OT10)T cells. Previous experiments demonstrated OT3 peptide has more affinity than OT10 peptide. Combined previous results of our laboratory, we chose the OT3 sequence as the CDR38 for the next studies.2 Retro virus-mediated yδTCR gene transfer into PBLs and evaluation of their functional activity against tumors.To improve the expression rate, we constructed TCRγ-andδ-chain gene retrovirus vectors. They were introduced into two individual vectors and generated TCR retrovirus vector particles. After 3 days activated, PBMC isolated from healthy volunteers were harvested, resuspended at 1 X 106 cells/ml inγδTCR retrovirus vector particles containing 200 IU/ml IL-2 and expressed yδTCR at the surface. The cells were called y982(OT3)T cells. After stimulated by various tumor protein extracts, the transfectant cells expressingγδTCR with OT3 sequence secreted much more TNF-a, IFN-y. At the same time,γ9δ2(OT3)T show the specific lysis against tumor cell lines and the lysis can be blocked by anti-γδTCR antibody. To study the mechanisms of the cytotoxocity, we incubated the y982(OT3)T cells with a FasL/Fas pathway inhibitor BFA or a perforin/granzyme inhibitor CMA at the different concentration before killing target cells. Results indicated that BFA partially blocked the killing ofγ9δ2(OT3)Tells against target cells (20-30%) and CMA suppressed the killing ability ofγ9δ2(OT3)T in Fashigh or Faslowcells, the inhibition rate was up to 33.93% and 56.71%, respectively. Combination CMA and BFA can mostly inhibit the killing process. These findings indicated that y982(OT3)T cells lysed their target cells via FasL/Fas and perforin/granzyme pathways, the latter was more important, especially in killing tumor cells that expressed low levels of Fas.3 Tumor cell therapy with y982(OT3)T combined with IL-2To observe anti-tumor effects in vivo, y982(OT3)T cells were used for experimental immunotherapy in nude mice engrafted with human ovarian cancer. Intratumoral injections of y9δ2(OT3)T cells greatly decreased the growth of tumors, significantly different from PBS control and vector retrovirus particle infected cells control following 18 days’therapy. Intratumoral injections of y9δ2(OT3)T cells prolonged the survival of tumor-bearing nude mice than the other two groups.In a word, we prepared gene-modified y8T cells using gene engineered technology. In vitro and vivo experiments, gene-modified y8T cells showed the anti-tumor potency, providing a new strategy for immunotherapy.Depression is the main type of mood disorders, characteristic with a significant and lasting decline in the mood. Depression has high disease incidence and mortality which threatens human health and life. The traditional theory that depression is a consequence of deficient monoamine activity has been revised by the addition of novel theories suggesting that disturbances in other systems are important for the pathophysiology of depression. In particular, there is evidence that glucocorticoids, cytokines and neurotrophins are involved in the manifestation and treatment of this disease. To date, the majority of studies on cytokines in depression have investigated in a single cytokine subset, especially monocytic proinflammatory cytokines such as IL-1β, IL-6 and TNF-a. However, cytokines exert pleiotropic activities and interact closely with each other. Also, individual cytokines belonging to the same subset may have different effects on the immune response. In the present study, we measured 3 subsets of cytokine family, specifically monocytic cytokines (IL-1β,IL-6,TNF-a), Thl cytokines (IFN-y,IL-2) and a Th2 cytokine(IL-4). The depressive patients showed a significant increase in serum levels of IL-1βand IL-6 compared to controls. And the IL-1βand IL-6 production were positively correlated(r=0.308 P<0.01), indicating IL-6 and IL-1βmay have a synergistic effect in the pathogenesis of depression. No significant differences in TNF-a level between both groups were found, although the mean values of this cytokines in MDD patients was slightly increased compared to that in the control subjects. In depressive patients, quantitative measurements were significantly decreased for IFN-y and IL-2 compared to those in controls. In contrast, serum levels of IL-4 were significantly higher in depressive patients, than that in control subjects. In addition, we tested glial cell line-derived neurotrophic factor (GDNF) and MICA expression in the serum of patients with depression. GDNF in patients with depression was significantly lower than that in the control group, and MICA was significantly higher than that of control subjects. Detection of these indicators of depression patients following the six weeks of fluoxetine treatment, there has been a marked restoration change. We believe that the level of GDNF and MICA may be one of the biological indicators. Of course, its etiology, pathological changes and other related mechanisms requires further study.In conclusion, in the second part of my thesis, we had studied the changes of cytokines, GDNA and MICA in serum of depressive patients, providing clues and basis for the further research on depression. |
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