白细胞介素-23基因修饰树突细胞获取凋亡癌细胞抗原诱导的抗胰腺癌免疫治疗

Therapeutic effects of treatment of pancreatic carcinoma by interleukin-23 and apoptotic cell antigen-modified dendritic cell vaccine: experiment with mice

目的探讨白细胞介素23(IL-23)基因转染的树突状细胞(DC)负载凋亡癌细胞抗原后诱导的免疫应答对小鼠胰腺癌的治疗作用。方法克隆并构建IL-23基因真核双表达载体,转染DC后检测其表型及自分泌IL-23和IL-12的能力;观察各组脾脏T淋巴细胞γ干扰素(IFN-γ)和IL-4的分泌量以及DC诱导的细胞毒T淋巴细胞(CTL)对胰腺癌细胞的杀伤作用。转染DC并负载肿瘤抗原后制备成疫苗对小鼠抗肿瘤的免疫保护作用和肿瘤抑制作用。结果基因测序证实IL-23基因克隆及双表达载体构建成功,转染后DC对共刺激分子MHC-Ⅰ和MHC-Ⅱ的表达增强;DC自分泌IL-23和IL-12的能力显著增强。DC介导的免疫应答促进了IFN-γ生成型Th1细胞的产生,IL-23转染组每24h IFN-γ的分泌(最高为535pg/ml/106)与其他各组比较(对照组每24h为60pg/ml/106),P<0.01;转染的DC疫苗在体内诱导出高水平的CTL活性(P<0.05)。接种IL-23修饰DC疫苗后小鼠的免疫防御能力显著增强;对肿瘤的生长有明显抑制作用,治疗组荷瘤鼠生存期与其他各组比较差异有显著意义。结论IL-23使DC抗原递呈能力更强,IL-23修饰DC疫苗可强化宿主针对特异肿瘤的CTL免疫应答,使宿主不仅产生防御性免疫反应而且增强自动免疫能力。

Objective To investigate the therapeutic effects on pancreatic carcinoma by dendritic cells （DCs） transfected with interleukin （IL）-23 and acquiring apoptotic cell antigen. Methods Murine IL-23 cDNA was subcloned into the vector so as to construct the eukaryotic dual-gene repression vector PeDNA3-IL-23. Bone-derived DCs were obtained from the long bones of extremities of normal Cs7 BL/6 mice and were transfected with pcDNA3-IL-23. DCs cultured routinely and IL-23-transfected DCs were cocultured with apoptotic tumor cells for 24 h so as to collect sensitized DCs acquiring the antigqan of the apoptotic cells. Primary pancreatic carcinoma models were created in Cs7 BL/6 mice with dimethyl-benzanthracene so as to obtain suspension of tumor cells. Thirty Cs7 BL/6 mice were randomly divided into 5 groups： Group Ⅰ （IL-23-transfected DC vaccine group, to be injected subcutaneously with IL-23 and apoptotic cells-modified DC vaccine twice with an interval of 7 d） , Group Ⅱ （apoptotic cells-sensitized DC vaccine group , to be injected subcutaneously with apoptotic cells-modified DC vaccine） , Group Ⅲ （IL-23 transfected DC group, to be injected with IL-23 transfected DCs）, Group IV （un-modified DC group, to be injected with un-modified DCs） , and Group Ⅴ （control group, injected with normal saline）. Seven days after the second injection suspension of tumor cells was injected subcutaneously, and then tumorigenesis was observed every other day. Another 30 C57 BL/6 mice were divided into 5 groups as above-mentioned （ Groups Ⅰ-Ⅴ, ） and were killed 1 week after the inoculation of tumor cell suspension. Their spleens were taken out. The cytotoxic lymphocytes （CTLs） were isolated, co-cultured with IL-25 and apoptotic cells, and then tested for the secretion of interferon （ IFN ） -γ and IL-4 by ELISA. Thirty C57 BL/6 mice loading pancreatic carcinoma were randomly divided into 5 groups as above-mentioned （ Groups Ⅰ-Ⅴ2 ） so as to observe the survival time. Results ELISA showed that the IFN-γ expression in the supernatant of culture fluid of IL-23 transfected DCs in Groups Ⅰ, was significantly up-regulated （ P 〈 0.01 ）, and the IL-4 secretion of Group Ⅰ, and Groups Ⅲ were both significantly lower than that of Group Ⅱ （ both P 〈 0. 05 ）. The CTL civilities of Group Ⅰ, and Ⅱ1 were both significantly higher than those of the other 3 groups （ all P 〈 0.05 ）. Four weeks after the inoculation tumorigenesis was not seen in Group Ⅰ and was seen in Groups Ⅲ and Ⅳ with an incidence of 33%-50%. Tumorigenesis occurred only 1 week later in Group Ⅴ. The tumor sizes of Groups Ⅰ2 and Ⅱ2 were both significantly smaller than those of the other 3 groups and the survival times of Groups Ⅰ2 and Ⅱ2 were both significantly lower than those of the other 3 groups （ all P 〈 0. 01 ）. Conclusion IL-23 helps enhance the presenting ability of DC antigen. IL-23 modified vaccine strengthens the immune response of CTLs against specific tumor, thus inducing defensive immune response in the host and strengthening their active immune ability.