吲哚胺2,3-双加氧酶在胰腺癌中的表达及其相关联合免疫治疗的效果研究

发布时间：2018-06-15 14:40

本文选题：胰腺癌 + 预后　；参考：《中国人民解放军医学院》2017年博士论文

【摘要】：研究背景及目的:胰腺癌恶性程度高,诊断困难,在目前中国恶性肿瘤致死率中排名第九,临床表现为快速的发展和转移。外科手术依然是胰腺癌的治疗标准,但多数患者在发现时已经因为远处转移和局部晚期肿瘤而成为手术禁忌。胰腺癌患者的五年生存率不足5%,即使经过根治性手术切除以后的患者,其五年生存率仍不足20%。肿瘤微环境中的免疫抑制是肿瘤生长,转移的关键因素,甚至可以诱导肿瘤的免疫逃逸。吲哚胺2,3-双加氧酶(IDO)是色氨酸经犬尿氨酸途径代谢的限速酶,具有免疫抑制效应,在多种肿瘤中表达并与预后相关。其在胰腺癌中的表达和作用目前仍没有报道。为研究IDO在胰腺癌中的预后意义以及可能的免疫治疗潜能,我们利用临床研究和基础实验相结合的方法分析IDO在胰腺癌发生发展中的作用和IDO相关免疫治疗的效果。方法:(1)采用Western Blot法和免疫组织化学法检测胰腺癌组织中的IDO表达量,并进一步分析IDO及临床数据与胰腺癌患者术后生存率的关系。(2)采用Real time PCR法和Western Blot法检测胰腺癌细胞系的IDO表达量和IFN-γ的调控作用,并分析体外条件下IDO抑制剂和AHR抑制剂的对胰腺癌细胞系的细胞毒性作用,为进一步体内动物实验奠定基础。(3)使用免疫健全小鼠构建皮下种植瘤模型,使用IDO抑制剂1-MT并联合免疫佐剂R848联合应用,观察其对胰腺癌的治疗效果,并使用流式细胞术分析肿瘤中免疫细胞数量。(4)使用抗体删除动物体内CD8+T细胞,并使用AHR抑制剂阻断AHR信号通路功能,观察各组肿瘤生长情况,评价其效果。分析1-MT联合R848治疗过程中肿瘤引流淋巴结中DC细胞的变化情况。结果:(1) IDO在胰腺癌中有表达,与肿瘤分化程度(P=0.017),TNM分期(P=0.03)和淋巴结转移情况(P=0.04)相关,而与年龄(P=0.87),性别(P=0.89),肿瘤大小(P=0.95),肿瘤位置(P=0.08)无关。并且胰腺癌组织中IDO的表达量(HR = 2.38 P =0.02)及淋巴结转移情况(HR = 2.86 P = 0.01)与胰腺癌患者预后相关,可以作为评价胰腺癌预后的独立指标。(2) IDO在胰腺癌细胞系中的表达,并且IFN-γ能够明显上调IDO的表达量;同时进行了 1-MT、CH223191、R848的体外细胞毒性实验,三种药物在体外条件下对细胞增殖无明显影响,可以用于胰腺癌动物模型免疫治疗相关实验。(3)建立胰腺癌皮下种植瘤模型,观察1-MT联合R848治疗胰腺癌效果,结果显示1-MT联合R848治疗组有较好的抑制肿瘤生长的效果,而单独使用同样剂量的1-MT和R848并不能很好的控制肿瘤的生长,进一步分析肿瘤中的免疫细胞,1-MT联合R848治疗组与对照组和R848组相比较,肿瘤中CD8+T细胞数目明显升高。(4) 1-MT和R848联合治疗组cDC细胞数目明显升高;CD8+T细胞删除组和CH223191+R848组,并不能抑制肿瘤生长。结论:胰腺癌组织中IDO的表达量与胰腺癌患者预后相关,并且IDO可以作为评价胰腺癌预后的独立指标;IDO在胰腺癌中的表达量受IFN- γ调控;1-MT和R848联合治疗抑制胰腺癌的效应是通过cDC提呈肿瘤抗原,活化T细胞,增加肿瘤中浸润CD8+T细胞来实现的,抑制IDO下游的AHR通路,并不能抑制肿瘤生长。
[Abstract]:Background and objective: pancreatic cancer is highly malignant and difficult to diagnose. It ranks ninth in the mortality rate of malignant tumor in China. The clinical manifestation is rapid development and metastasis. Surgery is still the standard of treatment for pancreatic cancer, but most of the patients have become contraindications to the operation because of distant metastasis and local advanced tumor. The five year survival rate of cancer patients is less than 5%. Even after radical resection, the five-year survival rate is still less than that of 20%. tumor microenvironment. The key factor of metastasis can induce tumor immune escape. The indolamine 2,3- dioxygenase (IDO) is the tryptophan through the canine urinary ammonia pathway. The rate limiting enzyme, which has the effect of immunosuppression, is expressed in a variety of tumors and is related to the prognosis. Its expression and role in pancreatic cancer are still not reported. To study the prognostic significance and potential immunotherapy potential of IDO in pancreatic cancer, we used a combination of clinical and basic testing to analyze the incidence of IDO in pancreatic cancer. The effect of the exhibition and the effect of IDO related immunotherapy. Methods: (1) the expression of IDO in pancreatic cancer tissues was detected by Western Blot and immunohistochemistry, and the relationship between IDO and clinical data and postoperative survival rate of pancreatic cancer patients was further analyzed. (2) the IDO table of pancreatic cancer cell lines was detected by Real time PCR method and Western Blot method. The effects of dose and IFN- gamma on the cytotoxicity of IDO inhibitors and AHR inhibitors on pancreatic cancer cell lines in vitro were analyzed. (3) the model of subcutaneous implants was constructed in immunized mice, combined with IDO inhibitor 1-MT and combined with immuno adjuvant R848. The therapeutic effect of pancreatic cancer and the use of flow cytometry to analyze the number of immune cells in the tumor. (4) the use of antibodies to delete CD8+T cells in animals, and the use of AHR inhibitors to block the function of AHR signal pathway, observe the growth of the tumor and evaluate the effect of the tumor, and analyze the changes of DC cells in the tumor drainage lymph nodes in the 1-MT combined with R848 treatment. Results: (1) IDO was expressed in pancreatic cancer, associated with tumor differentiation (P=0.017), TNM staging (P=0.03) and lymph node metastasis (P=0.04), but not related to age (P=0.87), sex (P=0.89), tumor size (P=0.95), and tumor location (P= 0.08). And the expression of IDO in pancreatic cancer tissues (HR = 2.38 P) and lymph node metastasis The condition (HR = 2.86 P = 0.01) is associated with the prognosis of pancreatic cancer patients. (2) the expression of IDO in the pancreatic cancer cell line, and IFN- gamma can obviously increase the expression of IDO; meanwhile, the cytotoxicity test of 1-MT, CH223191, R848 in vitro is carried out, and the three drugs can proliferate in vitro. No obvious influence, can be used in the animal model of pancreatic cancer immunotherapy related experiments. (3) to establish a model of pancreatic cancer subcutaneous implants and observe the effect of 1-MT combined with R848 in the treatment of pancreatic cancer. The results show that the combination of 1-MT and R848 has a better effect on inhibiting the growth of the tumor, and the same dose of 1-MT and R848 alone can not be well controlled. The tumor growth, further analysis of the tumor immune cells, 1-MT combined with R848 treatment group and the control group and the R848 group compared with the R848 group, the number of CD8+T cells increased significantly. (4) the number of cDC cells in the 1-MT and R848 combined treatment group increased significantly; CD8+T cell deletion group and CH223191+R848 group did not inhibit the tumor growth. Conclusion: I in the pancreatic cancer tissue, I. The expression of DO is associated with the prognosis of patients with pancreatic cancer, and IDO can be used as an independent index for evaluating the prognosis of pancreatic cancer. The expression of IDO in pancreatic cancer is regulated by IFN- gamma. The effect of 1-MT and R848 in the treatment of pancreatic cancer is achieved by presenting tumor antigen by cDC, activating T cells and adding CD8+T cells in the tumor to suppress IDO. The AHR pathway of swimming does not inhibit the growth of the tumor.
【学位授予单位】：中国人民解放军医学院
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R735.9

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