γδ T细胞亚群与乙肝相关肝细胞癌疾病进展和冷冻消融治疗预后关系研究

It is important to understand immunological factors that can predict the disease progression of HBV-related hepatocellular carcinoma (HCC) undergoing argon-helium cryoablation. Several lines of evidence indicate that human γδ T cells in livers are important contributors to innate immunity against cancer, but their regulatory role in controlling immune responses remains largely unknown. Recent studies demonstrated that Vδ2 T cells mediated antitumor immune response, however, its antitumor function was regulated by Vδ1 T cells through cell contact-independent and IL-4-mediated mechanisms in mice. In additon, Vδ1 T cells among lymphocytes in?ltrating breast tumors possessed a potent ability to suppress effector T cell responses and to block the maturation and function of antigen present cells, then lead to tumor escape. Our recent study found that γδ T cells, as well as Vδ2 T cells, are decreased in peripheral blood of HBV-associated liver cirrhosis and HCC patients. Analysis function of Vδ2 T cells documented that these cells exhibited impaired cytolytic activity, which closely associated with disease progression. As such, we hypothesize that tumor antigen or tumor microenvironment cause a profound decrease of Vδ2 T cells, which, in turn, lead to tumor escape during disease progression in HCC patients. Recently, we design studies to test the hypothesis, on one hand, through cross-sectional study and longitudinal study in a corhort of patients received cryoablation and then further confirm the findings in animal models, on the other hand, through optimizing the proliferation system of Vδ2 T cells.Our findings may provide novel evidences for the regulatory role of Vδ2 T cells in HCC, identify the potential role of Vδ2 T cells in predicting disease progression and facilitate the rational development of immunotherapeutic strategies for limiting or blocking hepatocellular carcinoma.

阐明肝细胞癌疾病进展和冷冻消融治疗过程中机体免疫应答规律，对在探讨肝癌发生发展免疫机制基础上建立肝癌综合治疗措施具有重要意义。γδ T细胞是肝内重要的抗肿瘤免疫细胞。小鼠模型发现，Vδ2 亚群是其抗肿瘤的关键效应细胞，其功能受Vδ1亚群调节。在人类乳腺癌，肿瘤内Vδ1亚群通过抑制多种免疫细胞促进肿瘤的免疫逃逸。我们前期结果显示，肝硬化和肝癌病人外周血γδT细胞显著降低，其中以Vδ2亚群数量和功能降低最为显著；且在晚期肝癌病人，Vδ2亚群的数量和功能降低程度更明显。因此，我们假设肝癌发生、发展过程中，肿瘤抗原或肿瘤微环境是造成Vδ2亚群数量和功能降低的重要原因，后者功能衰竭造成肿瘤免疫逃逸，促进肝癌的发生、发展。因此，我们建立体外Vδ2亚群扩增体系，结合冷冻消融治疗肝癌和动物模型证实上述假设。总之，本研究有利于阐明γδ T细胞及其亚群在肝癌疾病进展中的免疫致病机制，为今后临床应用提供基础。

结题摘要: 本课题历时4年，按照课题任务书和年度工作任务系统地开展了γδ T细胞亚群与乙肝相关肝细胞癌进展和冷冻消融治疗预后关系研究，取得重要结果如下：.1.明确乙肝相关肝癌、肝硬化和健康人外周血中γδ T细胞亚群的分布情况，以肝癌患者外周血中γδ T细胞频率和细胞数目最低，在肝癌患者中随着疾病进展γδ T细胞频率和细胞数目逐渐降低；Vδ2 T细胞亚群也表现出相似的趋势，但Vδ1 T细胞亚群在肝癌、肝硬化和健康人外周血中分布差异不明显；证实肝癌患者外周血Vδ2 T亚群Perforin和颗粒酶A、B表达显著降低；冷冻消融肝癌后肝癌患者外周血Vδ2 T亚群Perforin和颗粒酶A、B表达显著升高者预后好，认为抑制γδ T的数量及功能可能是肝细胞癌建立免疫逃逸的重要机制；冷冻消融后肝癌患者外周血中γδ T细胞（主要是v2亚群）的数量和频率较治疗前提高，以治疗后第7天明显增高者在28天时仍较治疗前的数量和分布频率增高的患者，其肿瘤局部控制率和生存期均好于冷冻消融后外周血中γδ T细胞（主要是v2亚群）的数量和频率较治疗前没有提高或降低的患者。.2.证实miRNA153在肝细胞癌小鼠模型中能够降低移植外源性γδ T细胞有效抑制动物模型中肿瘤生长的速度和大小；明确miR-153的稳定表达可导致PTEN缺陷，是肝癌细胞对细胞毒性化疗药物普遍耐药的关键，对指导开发新的肝细胞癌靶向药物具有重要的指导意义。.3.认为LINE-1 ORF-1p 是促进肿瘤细胞增值HGF/ETS-1 信号通路中一个新的共效应促进子；证实rhamnetin可通过下调Notch-1和Survivin的表达而促进内源性miR-34a的表达，而提高肝癌细胞对细胞性化疗药物和分子靶向药物的敏感性，延长肝癌患者治疗的临床应答率，对发展新的高效治疗肝细胞癌靶向药物有重要指导意义。