胰腺癌冷冻消融结合GM-CSF诱导自体原位肿瘤疫苗及有效肿瘤特异性抗原的筛选

The treatment of pancreatic cancer（PC） is very difficult. Residual tumor cell is the cause of recurrence and metastasis. The effective immunotherapy is one of the important way to remove residual tumor cells. Our previous researches showed that the cryoablation on the advanced PC was effective and it can induce anticancer immunity. Meanwhile, we found the the immune reponse after cryoablation was time-sensitive. For understanding the immunological mechanism induced by cryoablation furtherly and enhancing it's effect on pancreatic cancer, we will confirm the effect of the antigen from Ar-He cryoablation of tumor cell on the induction of the specific anti-cancer in PC. For searching the specific anti-PC antigen and making tumor vaccine, we will make situ tumor vaccine through combining the cryoablation and the GM-CSF and screen the specific tumor antigen after the cryoablation of PC with flow cytometry and X-ray single crystal diffraction technology and gasliquid chromatography and Proteomics related technology. Then the active of the tumor antigen will be identified. Furtherly, we will investigate the impact factors and the relation between the local and general immunity through observing the number change of immune cell in periperal blood and in the spleeen and the function change in the time and space distribution. Furthermore, we will assess the specific supression of tumor and assay the feasibility and effectiveness and time-sensitive. This study is to provide the evidence of the clinical treatment and new therapy modality on PC.

胰腺癌治疗后残存癌细胞是复发和转移根源，有效免疫治疗是清除残存癌细胞的重要手段。我们前期研究发现冷冻消融治疗中晚期胰腺癌效果明显，且可诱导抗肿瘤免疫；同时发现冷冻消融后免疫反应具有时效性。为深入了解冷冻后免疫反应及机制，提高胰腺癌治疗效果，拟通过动物实验进一步证实氩氦冷冻消融肿瘤细胞作为肿瘤抗原，诱导胰腺癌特异性免疫反应的有效性；通过冷冻联合GM-CSF方法诱导原位肿瘤疫苗，采用流式细胞术、X线单晶衍射技术、液相色谱分析、蛋白质组学等技术对冷冻消融后产生的肿瘤抗原进行筛选，以期寻找到胰腺癌特异性抗原，制备肿瘤疫苗；通过动态观察肿瘤局部、引流淋巴结及外周血和脾脏免疫细胞数量、功能在时间空间分布变化，探讨局部与全身之间内在联系及影响因素，评估疫苗对肿瘤的抑制作用，对胰腺癌防治的可行性、有效性及其时间－效应关系。旨在为胰腺癌提供临床治疗依据及新的治疗模式，具有重要临床意义和理论指导意义。

我们前期研究发现冷冻消融治疗中晚期胰腺癌效果明显，基础研究提示冷冻消融可能诱导抗肿瘤免疫，机制尚未明确，同时发现冷冻消融免疫反应具有时效性，如何增强冷冻消融后免疫防止肿瘤复发和转移值得关注。本研究拟通过体外实验证实，氩氦冷冻消融肿瘤细胞作为肿瘤抗原诱导胰腺癌特异性免疫反应的有效性。建立小鼠胰腺癌冷冻消融模型，在此基础上，联合（并选择）免疫调节剂GM-CSF诱导原位肿瘤疫苗。采用流式细胞术，X线单晶衍射技术，液相色谱分析，蛋白质组学相关技术对冷冻消融后产生的肿瘤抗原进行筛选，并鉴定其抗原活性，以期寻找到胰腺癌特异性抗原，制备肿瘤疫苗。. 研究先采用panc-1细胞系为研究对象，探讨胰腺癌细胞冷冻消融激活抗肿瘤免疫效应的有效性。将Panc-1细胞系分成实验组和对照组，实验组模拟临床进行冷冻消融，然后与DC细胞共培养；对照组直接同DC细胞共培养。分别检测两组INF-γ 的表达，MTT法检测两组T细胞对Panc-1细胞的杀伤力。结果：实验组IFN-γ的表达量明显高于对照组（P<0.001 ）。冻融后细胞激活的T细胞杀伤力显著大于对照组（P<0.001）。证实胰腺癌细胞冻融后可有效激活抗肿瘤免疫反应。. 将胰腺癌细胞冻融，DC细胞提取抗原，洗脱携带抗原的MHC，将抗原分类发现可能刺激抗肿瘤免疫的抗原分子集中为两条带，较为集中。研究将进一步分离提纯分析抗原氨基酸系列，确立抗原种类，验证其激活抗肿瘤免疫的有效性，最终制成肿瘤疫苗。

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