miRNA沉默信号调节蛋白活化树突状细胞诱导肝癌射频消融主动免疫的机制

The keypoint to improve the survivals of the patients with hepatocellular carcinoma (HCC) is to prevent the recurrence and metastasis after surgical resection. Immunotherapy is the most promising treatment method because it can discover and eradicate the tumor cells at any site. Previous studies have shown that signal regulatory protein alpha plays important suppressive roles in the stages of survial, maturation, migration,antigen-presenting for the DCs. In this project, in situ vaccination after radiofrequency ablation (RFA) for HCC, in combination with the miRNA silenced signal regulatory protein alpha to activating the dendritic cell(DC), is designed to achieve the strong specific active immune effect towards HCC.The combined method of in situ vaccination after RFA for HCC in mice and silence of signal regulatory protein alpha to activating the DCs will be established. The antitumor immune effect, immune protective effect, and antitumor specificity will be observed, including the growth of the existing tumor and the inoculated tumor, metastasis to the lung and lymph node, and the survivals of the rats.The antitumor mechanisms will be elucidated by observing the DC number, activation ratio, and phenotype in the tumor, spleen, and draning lymph node; the infiltration of T lymphocytes;the tumor-specific killing effect of CTL cells; T cell proliferation test; IFN-γ、IL-12、IL-4、IL-10 and Th1/Th2 in the peripheral blood. The project will establish a novel immunetherapy method for HCC with characteristics of simplicity, confirmed effect, and high safety. The novel treatment method will lower the recurrence and metastasis after resection of HCC and improve the patient survival.

肝癌术后复发和转移是影响患者生存的关键，免疫治疗因能发现和根除任何部位的肿瘤细胞成为最有希望的治疗模式。研究发现信号调节蛋白alpha在树突状细胞（DC）的生存、成熟、迁移、抗原递呈等阶段发挥了重要的抑制作用。采用射频消融联合miRNA沉默DC中的信号调节蛋白alpha的治疗方法，可能诱导强烈的抗肝癌主动免疫作用。项目将建立小鼠肝癌射频消融+miRNA沉默信号调节蛋白alpha活化DC的联合治疗方法；观察该方法的抗肿瘤免疫效应、免疫保护效应和抗肿瘤特异性，包括已存在肿瘤或再接种肿瘤的生长情况、肺及引流淋巴结转移、小鼠生存时间等；观察DC的数量、活化比例和表型变化，T淋巴细胞的浸润，IFN-γ分泌浓度、CTL细胞特异性杀伤活性、T细胞增殖试验、外周血IFN-γ、IL-12、IL-4、IL-10及Th1/Th2等指标阐明该方法抗肿瘤作用机制。项目有望建立一种肝癌免疫治疗新方法。

本研究旨在通过对DC中的SIRPα进行miRNA干扰，阻断SIRPα负向信号通路，进一步揭示SIRPα对DC和T细胞功能的影响，明确其在肝癌免疫治疗中的作用并分析其可能的分子机制。本研究通过慢病毒干扰技术有效抑制DC中SIRPα的表达，并通过体内体外实验证实SIRPα在DC生存、成熟和抗原递呈中起着重要的抑制作用。通过小鼠肿瘤预防模型进一步证实SIRPα在肿瘤免疫中发挥抑制性调节作用。干扰DC中SIRPα制备的DC疫苗可能成为肿瘤免疫治疗中重要的组成部分。另外我们通过实验证实了ITGB4在肝癌细胞中的表达显著上升。TGB4过表达后，能够明显促进肝癌细胞增殖、克隆形成和侵袭能力；干扰SLUG削弱细胞的增殖和迁移能力能够抑制EMT过程；抑制AKT/Sox2-Nanog信号通路。在小鼠肿瘤模型中进一步证实ITGB4可促进肿瘤生长及肺转移。ITGB4有望成为评估HCC的预后指标及HCC患者的治疗靶点。

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