目的：观察表达CD40L的小鼠结肠癌colon26瘤苗体内外对DC活性的影响。方法：以脂质体法将CD40L基因转染colon26细胞获得稳定表达CD40L的colon26/CD40L瘤苗。colon26/CD40L细胞与小鼠骨髓来源DC共培养，流式细胞术检测DC表型变化，RT-PCR法检测细胞因子基因P19、P35、P40、IL-18和IFN-γ的表达，ELISA法检测共培养上清中IL-12、IL-23和IFN-γ的水平。BALB/c小鼠皮下注射colon26细胞制备荷瘤小鼠模型，colon26/CD40L致敏DC治疗荷结肠癌小鼠，ELISA法检测荷瘤小鼠外周血中IL-12、IL-23、IFN-γ、IL-10和TGF-β的水平，H-E染色观察肝、脾和肿瘤组织的病理学变化。结果：成功获得高表达CD40L的丝裂霉素（MMC)处理的colon26/CD40L瘤苗。DC与colon26/CD40L瘤苗共培养后，DC表面共刺激分子CD80、CD86、MHCⅠ和MHCⅡ表达增高（P<0.01）；共培养体系中可检测到P19、P35、P40、IL-18和IFN-γ mRNA的表达，而在其他组未检测到上述基因的表达；共培养细胞上清中有较高水平的IL-12、IL-23和IFN-γ（P<0.01）。colon26/CD40L瘤苗致敏DC治疗组与colon26/CD40L瘤苗治疗组、DC治疗组比较，小鼠移植瘤的体积和质量明显减小和减少（P<0.05）；小鼠外周血IL-12、IL-23和IFN-γ明显升高（P<0.01），IL-10和TGF-β明显降低（P<0.01）；小鼠移植瘤组织病理变化显著减轻。结论：表达CD40L的丝裂霉素（MMC)处理的结肠癌瘤苗可促进DC的成熟，刺激DC分泌细胞因子，增强DC体内外活性，从而增强治疗体系的抗肿瘤免疫效应。

Objective:To explore the in vitro and in vivo effects of CD40L-expressing murine colon cancer colon26 vaccine on activities of dendritic cells (DCs). Methods: The CD40L gene was transfected into colon26 cells using lipofactamine method, and the stable colon26/CD40L transfectants were obtained. Colon26/CD40L cells were co-cultured with DCs, and the phenotype of DCs was examined by flow cytometry; the expressions of cytokine genes such as IL-12, IL-23, IL-18 and IFN-γ were examined by RT-PCR; and the IL-12, IL-23 and IFN-γ levels in co-cultured supernatants were measured by ELISA. Tumor-bearing BALB/c mouse model was prepared by subcutaneous injection of colon26 cells, and treated with DCs impulsed with colon26/CD40L vaccine. IL-12, IL-23, IFN-γ, IL-10 and TGF-β levels in mouse peripheral blood were detected by ELISA; and the histopathological changes of the liver, spleen and tumor tissues were observed by H-E staining. Results: Colon26 cells stably expressing CD40L (colon26/CD40L vaccine) were successfully obtained. DCs, when co-cultured with colon26/CD40L vaccine, had up-regulated expressions of co-stimulatory molecules, including CD80, CD86, MHCⅠ and MHCⅡ (P<0.01). The expressions of IL-12, IL-23, IL-18 and IFN-γ mRNA genes were only detected in DC-colon26/CD40L system, not in other co-cultured systems. And the DC-colon26/CD40L co-cultured system showed higher IL-12, IL-23 and IFN-γ levels (P<0.01). Compared with mice treated with colon26/CD40L or DCs, mice treated with DCs-colon26/CD40L had smaller tumor volumes and lower weights (P<0.05), increased IL-12, IL-23, and IFN-γ levels in the peripheral blood (P<0.01), decreased IL-10 and TGF-β levels (P<001), and better histopathological changes of tumor tissues. Conclusion: CD40L-expressing colon cancer cells can promote the maturation, induce the cytokine secretion, and enhance the activities of DCs in vitro and in vivo.

河北省科技厅科技计划项目(No.09276101D-29);河北省高校强势特色学科项目(No.2006-2010)