硝呋齐特增强负载肿瘤细胞裂解物DC疫苗抗肝癌效应研究

Hepatocellular carcinoma (HCC) is a highly aggressive malignancy with poor prognosis and high mortality. At present, vaccination with dendritic cells (DC) pulsed with tumor cell lysate has been proved that it is an effective therapy against HCC. However, after the immunization with this vaccine, the ability of promoting the specific T cell immune response is rather weak, influencing the anti-tumor response induced by DC vaccine. Thus, it is necessary to research an effective adjuvant to improve the anti-tumor effect of DC. Activation of signal transducer and activator of transcription 3 (Stat3) significantly inhibited the immune response of anti-tumor, DC maturity and anti-tumor response of CD8+ CTL, increase the number of Treg in tumor. Applicant has participated to publish a paper that pointed out inhibited the Stat3 activation could improve the anti-tumor immune response, increase the immune cells infiltration in tumor-tissue and inhibit the growth of HCC in the mouse model of orthotopically implanted HCC. Nifuroxazide, the antidiarrheal agent, has been proved to directly inhibit STAT3. Thus, we will confirm whether nifuroxazide could improve the anti-tumor immune response in mice vaccination with DC pulsed with tumor cell lysate. The study will provide the theoretical and experimental basis on the effectual adjuvant for the DC vaccine to treat HCC.

肝癌除早期手术外尚无有效治疗手段。负载肿瘤细胞裂解物的DC疫苗治疗肝癌已应用于临床并取得一定疗效。然而其诱导机体特异性T细胞免疫应答能力较弱，影响了这种疫苗的抗肿瘤作用。因此，需要寻找一种安全有效的佐剂来提高DC疫苗诱导的抗肿瘤免疫反应。 研究显示，Stat3基因的激活通过影响DC成熟抑制CD8+ CTL抗肿瘤作用，通过促进Treg细胞在肿瘤内积聚，抑制机体抗肿瘤免疫应答。申请者研究证实，抑制机体Stat3表达能明显增强机体抗肿瘤免疫反应，增加肿瘤微环境中免疫细胞浸润，抑制小鼠肝癌生长。硝呋齐特是一治疗胃肠道感染药物，近年被证实能够直接抑制Stat3表达，但其用于肿瘤相关治疗尚未见报道。本课题拟通过体内外实验研究硝呋齐特作为佐剂增强DC疫苗治疗肝癌的免疫效果及其机制，为临床应用提供依据。

目前，负载肿瘤细胞裂解物的DC疫苗治疗肝癌已经取得一定的疗效。然而这种疫苗对于诱导机体的CD4+及CD8+T细胞分别向Th1及CTL分化的能力较弱，影响了机体抗肿瘤免疫应答及DC疫苗的抗肿瘤作用。因此，寻找一种安全有效的佐剂来提高DC疫苗诱导的抗肿瘤免疫反应非常重要。研究显示，Stat3基因的激活会通过抑制DC成熟、CD8+ CTL细胞抗肿瘤作用，抑制机体抗肿瘤免疫应答。Nifuroxazide做为治疗胃肠道感染的药物，已被证实能够直接抑制Stat3表达，但其对于机体抗肿瘤的免疫增强作用尚未见报道。因此，本课题将研究Nifuroxazide对于负载肝癌细胞裂解物的DC疫苗治疗肝癌的免疫增强作用。. 我们在小鼠肝原位癌模型建立后第7天，随机将小鼠分为四组：PBS组、nifuroxazide组、DC疫苗组及联合组。记录小鼠的生存率及肿瘤大小。同时检测肿瘤组织中CD4+、CD8+T淋巴细胞的浸润、小鼠脾脏中CD4+、CD8+ T细胞及NK细胞的比率、小鼠血清中IFNγ、TNFα及VEGF的含量、肿瘤组织中与迁移及凋亡相关蛋白的表达。. 我们的结果显示，与对照组相比，联合应用Nifuroxazide与负载TCL的DC疫苗能够有效地提高荷瘤小鼠的生存率。在接种肿瘤后40天，模型组小鼠的生存率为25%，而nifuroxazide（200μg/只）组为50%，DC疫苗组为33.3%，而nifuroxazide联合负载TCL的DC疫苗组的生存率为58.3%；提高了荷瘤小鼠脾脏中CD4+及CD8+ T淋巴细胞的表达，增加了肿瘤组织中CD4+及CD8+ T淋巴细胞的浸润，提高了小鼠血清中IFNγ及TNFα的含量，降低了VEGF的含量。不仅如此，联合应用Nifuroxazide与负载TCL的DC疫苗显著抑制了p-stat3及MMP2的表达，增加了cleaved-PARP及cleaved-caspase3的表达。. 该项目为临床上提高DC疫苗治疗肿瘤的效果提供了一定的理论及实验依据。