【作者】 李东印； 孙桂森； 段东明； 张建智； 闵军； 褚忠华； 谷川；

【Author】 Li Dong-Yin Sun Gui-Sen Duan Dong-Ming Zhang Jian-Zhi Min Jun Chu Zhong-Hua GuChuan Department of General Surgery, First central hospital of Tianjin, Tianjin, 300192,China Department of Hepatic-Biliary Surgery, The Second Affiliated Hospital of Sun Yat-sen University, Guangzhou, 510120, Chian

【机构】 天津市第一中心医院普外科； 中山大学附属第二医院肝胆外科；

【摘要】 目的:肿瘤的生物治疗正成为肿瘤学研究的热点, 并被称为继手术、化疗、放疗之后的第四种治疗模式,随着对树突状细胞(dendritic cells,DCs)在免疫反应中所起的中心作用的认识的加深,以及体外大量培养DCs方法的建立, 以DCs为平台的肿瘤生物治疗受到广泛关注。树突状细胞是目前发现功能最强的专职抗原呈递细胞(APC),在体外制备负载肿瘤抗原的肿瘤特异性的DCs瘤苗,再注射回体内以激发机体产生主动性抗肿瘤免疫反应,是具有良好前景的生物治疗方案,目前已应用多种肿瘤的临床治疗。本研究的目的是制备肿瘤裂解物负载的DCs瘤苗并观察其体外诱导抗肿瘤免疫效应,探讨其制备方法。方法:抽取健康人(n=4)外周血, 以Ficoll密度梯度离心法分离外周血单个核细胞(peripheral blood mononuclear cells,PBMCs),以细胞因子GM—CSF 和IL-4在体外将其诱导为树突状细胞,第6天采用不同方法分组处理:A组,仅用GM—CSF 和IL-4培养; B组,负载人肝癌细胞冻融抗原,C组,联合负载抗原及以TNF-α诱导成熟;D组,联合负载抗原及以鸡尾酒法(TNF-α+ IL-6+IL-1β+PGE2)诱导成熟。24小时后收获各组DCs以流式细胞仪检测其成熟表型CD80、CD83、CD86和LHA- DR,以ELISA法检测其IL-12的分泌,MTT法检测其刺激淋巴细胞增殖活性,将树突状细胞瘤苗与淋巴细胞以1:10的比例共培养获得免疫效应细胞,以乳酸脱氢酶释放试验检测免疫效应细胞对肝癌细胞的特异性细胞毒作用。结果:抗原负载或联合细胞因子均可诱导DCs的成熟和IL—12的分泌 (P<0．05),成熟的DCs有较强的刺激淋巴细胞增殖能力;抗原负载的B组、C组和D组DCs可诱导效应细胞对肝癌细胞 BeL-7402的特异性杀伤作用,以D组最为明显(P<0．05)。结论:抗原负载的DCs可体外诱导特异性抗肿瘤免疫效应, 提示临床上以抗原负载的树突状细胞作为肿瘤免疫治疗的方法是可行的。更多还原

【Abstract】 Objective: Biotherapy or immunotherapy of cancer becomes a focus of oncology study, and have a brilliant prospects in the clinical application. Immunotherapy is now considered a fourth modality of cancer treatment besides of operation, chemotherapy and radiotherapy. Recently, dendritic cells is becoming increas- ingly recognized as pivotal in initiating effective immune responses against tumors. The methods for the large scale ex vivo generation of DCs from peripheral blood mono-cytes had been established. Application of DCs as a tumor vaccine for immunotherapy has been becoming an attractive field in tumor treatment. Dendritic cells is the most potent ’professional’ antigen presenting cells. Ex vivo generation of autologous antigen-loading DCs, followed by injection of DCs for the in vivo generation or boosting of antigen-specific T cell mediated active antitumor immunity has been the most common method and represents an attractive anticancer strategy to date. Numerous DCs based clinical trials have been performed for a wide range of tumors. The objective of this study is to investigate the antitumor immunity effect of dendritic cells after loading antigen and its preparation methods in vitro. Methods: The peripheral blood mononuclear cells (PBMC) were isolated from venous blood samples of healthy donors (n=4) by Ficoll method. The human monocytes-derived dendritic cells were induced in the presence of cytokine GM-CSF and IL-4 and divided into four groups after six days, group A, only in presence of GM-CSF and IL-4, group B, antigen loading with tumor cell lysate, group C, combination of antigen loading and TNF- α maturation induction, group D, combination of antigen loading and matured by cytokine cocktail (TNF- α + IL-6+IL-1 β +PGE2 X The maturation surface markers of CD80, CD83, CD86 and HLA-DR were detected by FCM and IL-12 production was detected by ELISA respectively after 24 hours. The T cells stimulatory proliferation capacity was measured by MTT. The effect lymphocytes were generated by co-culture the DCs and autologous T lymphocytes at the rate of 1:10. The specific cytotoxicity of T cells was measured by LDH release assay. Results: The DCs maturation and IL-12 production could be both induced by antigen loading or combination with cytokines (P<0.05 ). The T cells stimulatory proliferation capacity were increased by mature DCs. The antigen loading DCs, group B , C and D, could induce the specific cytotoxicity of T cells to BeL-7402 hepatoma cells in vitro and the group D showed highest induction capacity ( P<0.05 ). Conclusion: The antigen loading DCs can induce the specific anti-tumor immunity in vitro. It’ s suggested that the antigen loading DCs as an anti-tumor immunotherapy strategie are suitable for clinical application.更多还原