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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

发布时间:2018-07-29 18:42
【摘要】:射频消融(Radiofrequency ablation,RFA)是目前广泛应用于临床的微创治疗手段。其不仅通过物理热效应直接损毁肿瘤,而且消融使肿瘤抗原暴露,刺激机体产生免疫反应。然而,射频消融引起的抗肿瘤免疫反应并不足以阻止肿瘤的复发及转移。细胞因子诱导的杀伤(Cytokine-induced killer,CIK)细胞是外周血单个核细胞在体外经有序添加多种细胞因子(IL-2、抗CD3单克隆抗体、IFN-γ等)培养后获得的一组异质性细胞群。其兼有T淋巴细胞的杀瘤活性和自然杀伤细胞样非组织相容性复合物(MHC)限制性的抗瘤特点。然而,在实体瘤治疗方面,CIK细胞治疗仍然处于辅助地位,单独应用往往疗效不佳。影响CIK细胞在体内发挥作用的一个重要因素是:效应性免疫细胞能否到达靶器官,实现与肿瘤细胞的直接接触,从而有效杀伤肿瘤细胞。据此,理论上,在消融治疗后的恰当时间内输注CIK细胞,消融产生的免疫刺激可能促进CIK细胞向肿瘤部位迁移、定植,增强CIK细胞的体内抗肿瘤效应。同时,CIK细胞在肿瘤部位聚集又可放大消融诱发的抗肿瘤免疫反应,两者相互增效产生最大的协同治疗效能。本课题从动物实验到临床试验两部分探讨射频消融联合CIK细胞的协同抗肿瘤效应。第一部分射频消融与CIK细胞协同抗肿瘤效应的动物实验研究目的观察RFA联合CIK细胞的协同抗肿瘤作用,探讨RFA对CIK细胞体内迁移及杀瘤活性的影响及可能作用机制。方法1、建立小鼠双侧背部B16黑色素瘤与CT26结肠癌皮下移植瘤模型,利用荷瘤鼠脾脏细胞培养CIK细胞,检测CIK细胞表面趋化因子受体的表达。2、成瘤小鼠分四组:单独右侧肿瘤RFA治疗组、单独CIK治疗组、两者联合治疗组及未治疗对照组,记录未消融侧肿瘤大小与小鼠生存时间,绘制生存曲线。3、采用流式细胞术分析各治疗组未消融侧肿瘤内淋巴细胞亚群比例与数量、免疫抑制性细胞群:调节性T细胞(T regulatory cell,Treg),髓源性抑制细胞(Myeloid-derived suppressor cells,MDSC)的比例。4、利用不同免疫表型小鼠检测消融后浸润至未消融侧肿瘤内外源性CIK细胞的数量与活性。5、采用ELISA法及Real Time-PCR技术检测单纯消融后1、3、6、9、12天未消融侧肿瘤内趋化因子CXCL10的表达,利用Cxcl10KO小鼠检测CXCL10在RFA激发抗肿瘤免疫反应,促进CIK细胞迁移中的作用。6、评估RFA与CIK细胞输注时间间隔对治疗效果的影响。结果1、成功培养CIK细胞,高表达趋化因子受体CXCR3、CXCR4、CCR5。2、RFA与CIK单独治疗均可产生短暂的抑制肿瘤生长作用,而联合治疗能显著抑制未消融侧肿瘤生长;联合治疗组生存期为42±3.3d,显著长于未治疗对照组(20.5±2.2d)、单独右侧肿瘤RFA治疗组(26±2.2d)及单独CIK治疗组(31.5±2.2d)(P0.001)。3、联合治疗组小鼠未消融侧肿瘤内淋巴细胞浸润增加,其中CD8+、CD4+T、NK(CD3-NK1.1+)和NKT(CD3+NK1.1+)细胞数量均显著增加;CD8+/Treg(CD4+Fox P3+)比值增大;MDSC比例降低。4、RFA联合CIK组较单独CIK组小鼠未消融侧肿瘤内外源性CIK细胞聚集增加,并且外源性CIK细胞Ki-67、可诱导共刺激分子(Inducible costimulator,ICOS)与颗粒酶B(Granzyme B)的表达水平较未RFA组明显上调。5、RFA动态上调远处肿瘤组织CXCL10,CXCL10基因敲除减弱RFA激发的抗肿瘤免疫应答,影响CIK细胞的迁移。6、RFA后3天输注CIK细胞能产生协同抗肿瘤效应,12天后则无明显协同效应。结论1、RFA联合CIK细胞治疗可增强消融区外肿瘤微环境中抗肿瘤免疫反应。2、RFA治疗可促进外源性CIK细胞向消融区外肿瘤内迁移,提高CIK细胞在肿瘤内的增殖能力与杀瘤活性,二者联用具有协同抗肿瘤作用。3、RFA激发的抗肿瘤免疫反应和CIK细胞的迁移依赖于趋化因子CXCL10。4、RFA与CIK细胞的输注时间间隔影响疗效,应尽早输注,这对指导两者联合的临床应用具有重要意义。第二部分射频消融联合CIK细胞治疗结直肠癌肝转移瘤的临床研究目的观察RFA联合静脉输注CIK细胞治疗结直肠癌术后单纯肝转移患者的有效性和安全性,评估二者联合应用对机体肿瘤抗原特异性免疫能力的影响。方法入组结直肠癌术后单纯肝转移患者60例,经多学科团队讨论,签署知情同意书后分为单纯消融组(RFA):对肝脏转移瘤消融,不行CIK细胞治疗。消融联合CIK细胞治疗组(RFA+CIK):消融前7天,分离患者外周血单个核细胞(PBMC),体外培养CIK细胞,肝转移瘤消融后7天输注体外培养好的CIK细胞。比较两组患者的无疾病进展时间(PFS)、总生存时间(OS)、不良反应。ELISPOT法检测RFA+CIK组中外周血CEA50ng/ml的患者消融前、消融后7天(CIK细胞治疗前)、消融后14天(CIK细胞治疗后)三个时间点,PBMC在CEA抗原肽刺激下分泌IFN-γ的能力。结果RFA+CIK组和RFA组中位PFS分别是23个月、18个月,2年PFS率分别为41.9%和26.7%,3年PFS率分别为20.3%和13.3%,差异具有统计学意义(P=0.0336)。RFA组的中位OS为43个月,RFA+CIK组目前还在随访中。RFA组3年OS率为64.6%,而RFA+CIK组3年OS率为81%,但差异无统计学意义(P=0.1187)。ELISPOT试验发现RFA+CIK组,8名外周血CEA50ng/ml的患者中,消融后7天(CIK细胞治疗前)有6名患者PBMC中具有分泌IFN-g能力的细胞数量较消融前增加(P=0.010);而在消融后14天(CIK细胞治疗后),有7名患者PBMC中具有分泌IFN-γ能力的细胞数量较消融后7天(CIK细胞治疗前)增加(P=0.028)。这8名患者在消融联合CIK细胞治疗后PBMC中具有分泌IFN-γ能力的细胞数量均较治疗前增加(P=0.001),差异有统计学意义。结论RFA联合CIK细胞治疗可增强结直肠癌肝转移患者肿瘤抗原特异性免疫应答,延长疾病进展时间(PFS),具有协同抗肿瘤效应。综上所述,本研究通过动物实验和临床试验两部分探讨RFA联合CIK细胞的协同抗肿瘤效应。动物实验建立双侧背部荷瘤小鼠模型,利用荷瘤鼠脾脏细胞制备CIK细胞,临床研究入组结直肠癌术后单纯肝转移患者。本研究明确了:1)RFA联合CIK细胞治疗可增强消融区外肿瘤微环境中抗肿瘤免疫反应;2)RFA治疗可促进外源性CIK细胞向消融区外肿瘤内迁移,增强CIK细胞在肿瘤内的增殖能力与杀瘤活性;3)趋化因子CXCL10在RFA激活的抗肿瘤免疫应答及CIK细胞的迁移中发挥重要作用;4)RFA与CIK细胞的输注时间间隔影响疗效,应尽早输注CIK细胞。5)RFA联合CIK细胞治疗可增强结直肠癌肝转移患者肿瘤抗原特异性免疫应答,延长疾病进展时间(PFS)。二者联用具有协同抗肿瘤效应。
[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

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