射频消融与细胞因子诱导的杀伤细胞协同抗肿瘤效应的研究
[Abstract]:Radiofrequency ablation (RFA), which is widely used in clinical minimally invasive treatment, not only directly damage the tumor by physical heat effect, but also exposes the tumor antigen to stimulate the body to produce immune response. However, the immune response to the anti swelling tumor caused by radiofrequency ablation is not enough to prevent the recurrence and metastasis of the tumor. Cytokine induced cytotoxic (Cytokine-induced killer, CIK) cells are a group of heterogeneous cells obtained from peripheral blood mononuclear cells cultured in vitro after a variety of cytokines (IL-2, anti CD3 monoclonal antibodies, IFN- gamma, etc.). The cytotoxic activity of T lymphocytes and the natural killer cell like non tissue compatibility complex are also obtained. However, CIK cell therapy is still in an auxiliary position in the treatment of solid tumors, and the effect of individual application is often poor. One of the important factors that affect the role of CIK cells in the body is whether the effector cells can reach the target organs and achieve direct contact with the tumor cells, thus effectively killing the CIK. Therefore, in theory, CIK cells are transfused within the appropriate time after the ablation treatment. The immune stimulation produced by the ablation may promote the migration of CIK cells to the tumor site, colonize and enhance the anti tumor effect of CIK cells in vivo. At the same time, the aggregation of CIK cells at the tumor site can also increase the anti-tumor immune response induced by large ablation. Both of them increase each other. The maximum synergistic efficacy was produced. In this subject, the synergistic antitumor effect of radiofrequency ablation combined with CIK cells was investigated from two parts of animal experiment to clinical trial. Part 1 animal experimental study on the synergistic anti tumor effect of radiofrequency ablation and CIK cells was studied in order to observe the synergistic antitumor effect of RFA combined with CIK cells, and to explore the RFA to CIK cells. Effect and possible mechanism of tumor activity in vivo and in vivo. Methods 1, a mouse model of subcutaneous transplantation of B16 melanoma on the bilateral back and CT26 colon cancer was established. The expression of CIK cells in the spleen cells of the tumor mice was used to detect the expression of.2 in the CIK cell surface chemokine receptor, and the tumor mice were divided into four groups: the alone right tumor RFA treatment group, and CIK alone CIK. The treatment group, both combined treatment group and untreated control group, recorded the size of unablative side tumor and the survival time of mice, and plotted survival curve.3. Flow cytometry was used to analyze the proportion and quantity of lymphocyte subsets in the unablative side tumor of each treatment group, and the immunosuppressive cell group: regulatory T cells (T regulatory cell, Treg), myelinated inhibition. The proportion of Myeloid-derived suppressor cells (MDSC) was.4, and the number and active.5 of the endogenous and external CIK cells infiltrated to the non ablation side tumor were detected by different immunophenotype mice. The expression of chemokine CXCL10 was detected by ELISA method and Real Time-PCR technique in the non ablation side tumor after pure ablation. Cxcl10KO mice detected CXCL10 in RFA to stimulate the anti-tumor immune response and promote the role of CIK cell migration,.6, to evaluate the effect of the time interval between RFA and CIK cells. Results 1, the successful culture of CIK cells, high expression of chemokine receptor CXCR3, CXCR4, CCR5.2, RFA and solitary therapy can produce a brief inhibition of tumor growth. Combined therapy could significantly inhibit the growth of unablative side tumors; the survival period of the combined treatment group was 42 + 3.3D, significantly longer than that in the untreated control group (20.5 + 2.2d), the individual right tumor RFA treatment group (26 + 2.2d) and the single CIK treatment group (31.5 + 2.2d) (P0.001).3, and the increase of lymphocyte infiltration in the unablative side tumor of the combined treatment group, of which CD8+ was in the combined treatment group. The number of CD4+T, NK (CD3-NK1.1+) and NKT (CD3+NK1.1+) cells increased significantly, the ratio of CD8+/Treg (CD4+Fox P3+) increased, the proportion of MDSC decreased.4, RFA joint CIK group increased the aggregation of endogenous and external tumor cells in the non ablation side tumor of mice. The expression level of granulase B (Granzyme B) was significantly up-regulated than that in the non RFA group, and RFA dynamically up-regulated the CXCL10 in the distant tumor tissue. CXCL10 gene knockout weakened the anti tumor immune response of RFA stimulated and influenced the.6 migration of CIK cells. 3 days after RFA, the transfused cells could produce synergistic antitumor effects and no obvious synergistic effect in 12 days. Conclusion 1 Cell therapy can enhance the anti tumor immune response.2 in the tumor microenvironment outside the ablation area. RFA therapy can promote the migration of exogenous CIK cells into the tumor outside the ablation area, improve the proliferation and tumor activity of CIK cells in the tumor. The two combined anti-tumor immune response and CIK cell migration dependence with.3, RFA excitation and CIK cells are combined. The time interval between chemokines CXCL10.4, RFA and CIK cells affects the curative effect and should be delivered as early as possible. This is of great significance for guiding the combination of the two clinical applications. Second parts of the clinical study on the combination of radiofrequency ablation combined with CIK cells in the treatment of colorectal cancer liver metastases; RFA combined intravenous infusion of CIK cells for the treatment of colorectal cancer The effectiveness and safety of the patients with simple liver metastasis were evaluated by the combined application of two patients with tumor antigen specific immunity. Methods 60 patients with simple liver metastasis after colorectal cancer surgery were discussed by a multidisciplinary team. After signing informed consent books, the patients were divided into simple ablation group (RFA): liver metastases ablation, and no CIK cell treatment Treatment. Ablation combined with CIK cell therapy group (RFA+CIK): 7 days before the ablation, the peripheral blood mononuclear cells (PBMC) were isolated, CIK cells were cultured in vitro, and the CIK cells were transfused for 7 days after the liver metastases were ablated. The time of disease progression (PFS), total survival time (OS) and adverse reaction.ELISPOT method were compared between the two groups and the RFA+CIK group. Before ablation of blood CEA50ng/ml, 7 days after ablation (CIK cell therapy), 14 days after ablation (after CIK cell therapy), the ability of PBMC to secrete IFN- gamma under the stimulation of CEA antigen peptide. Results the PFS in group RFA+CIK and RFA group was 23 months, 18 months, 2 years, 41.9% and 26.7% respectively, and 20.3% and 13.3% respectively in 3 years, respectively, and the difference was 20.3% and 13.3% respectively. The difference was 20.3% and 13.3% respectively. The median OS in group.RFA was 43 months, and the RFA+CIK group was still followed up with the OS rate of 64.6% in the.RFA group and 81% in the RFA+CIK group for 3 years, but the difference was not statistically significant (P=0.1187) in the RFA+CIK group and 8 of the 8 peripheral blood CEA50ng/ml. The 7 days after the ablation (before the treatment) there were 6 patients. The number of cells with the ability to secrete IFN-g increased in C (P=0.010), and in 7 patients, the number of cells with the ability to secrete IFN- gamma in PBMC increased (P=0.028) at 14 days after ablation (after CIK cell therapy). These 8 patients secreted IFN- gamma ability in PBMC after ablation combined with CIK cell therapy. The number of cells increased in comparison with that before treatment (P=0.001), and the difference was statistically significant. Conclusion RFA combined with CIK cell therapy can enhance the specific immune response of patients with colorectal cancer liver metastasis, prolong the time of disease progression (PFS) and have synergistic antitumor effect. In summary, two parts of animal and clinical trials are discussed in this study. The synergistic anti-tumor effect of RFA combined with CIK cells. In animal experiments, a mouse model of bilateral back bearing tumor was established, and CIK cells were prepared by using the spleen cells of the tumor bearing mice. The clinical study was conducted in a group of patients with simple liver metastasis after the operation of rectal cancer. 1) RFA combined with CIK cell therapy could enhance the anti tumor immune response in the microenvironment of the ablation area. 2) RFA therapy can promote the migration of exogenous CIK cells into the tumor outside the ablation area, enhance the proliferation and tumor activity of CIK cells in the tumor; 3) chemokine CXCL10 plays an important role in the anti-tumor immune response activated by RFA and the migration of CIK cells; 4) the interval between the infusion time of RFA and CIK cells affects the curative effect, and the CIK fine should be injected as soon as possible. Cell.5) RFA combined with CIK cell therapy can enhance the specific immune response of cancer antigen in patients with colorectal cancer and prolong the time of disease progression (PFS). The combination of the two has a synergistic antitumor effect.
【学位授予单位】:苏州大学
【学位级别】:博士
【学位授予年份】:2016
【分类号】:R730.5
【参考文献】
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