目的：探讨重组腺病毒Ad5-CCL20联合热疗对结肠癌CT-26细胞小鼠移植瘤的抑制作用。方法:将Ad5-CCL20转染CT-26细胞，采用ELISA法和趋化实验分别检测CT-26细胞中CCL20的表达及其对DC的趋化作用。Western blotting检测热激结肠癌CT-26细胞中HSP70、GP96的表达。用热激后细胞蛋白（Heat组）对DC进行诱导，并设对照组（Control组）和阴性对照组（Unheat组），流式细胞术检测DC的表型。建立CT-26细胞移植瘤模型，设Ad5-CCL20/Heat、Heat、Ad5-CCL20、Ad5-GFP和Control组，观察各组荷瘤小鼠肿瘤生长和生存时间；采用ELISAPOT法和LDH法分别检测脾组织T淋巴细胞分泌IFN-γ的能力和对CT-26细胞的CTL杀伤活性，免疫组化法检测肿瘤组织DC浸润情况。结果：Ad5-CCL20转染CT-26细胞后可高表达CCL20，且对iDC、mDC都具有趋化活性，对iDC更为明显（P<0.05）。CT-26细胞热激后可高表达诱导型HSP70和GP96。与Control、Unheat组相比，Heat组细胞蛋白诱导后DC的CD80、CD86、CCR6和MHC-Ⅱ的表达率明显升高（均P<0.01）。与Control、Ad5-GFP、Ad5-CCL20组和Heat组相比，联合治疗组荷瘤小鼠的肿瘤抑制最为明显(均P<0.01)，生存时间明显延长（均P<0.05）。联合治疗后的荷瘤小鼠脾组织T淋巴细胞的CTL活性要明显高于另外四组（均P<005），其瘤组织中CD11c+ DC的阳性率也明显增高（均P<0.05）。结论：重组腺病毒Ad5-CCL20联合热疗可召募并促进DC成熟和抗原提呈，明显抑制肿瘤生长，并延长荷瘤小鼠的生存期，提示其对结肠癌有潜在的治疗作用。

Objective:To explore the inhibitory effect of recombinant adenovirus Ad5-CCL20 combined with hyperthermia on the growth of mouse colon carcinoma CT-26 cells in mice transplanted tumor.Methods: Colon carcinoma CT-26 cells were transfected with recombinant adenovirus Ad5-CCL20, the expression of chemokine CCL20 was assessed by ELISA method and its chemotaxis to dendritic cell(DC) was detected by chemotaxis assays.Western blotting was performed to detect the expression of HSP70 and GP96 after CT-26 cells heated. DC were induced with protein exosomes from heat-shock CT-26 cells(heat group), control group and negative group as controls. Phenotypes of DC were detected by flow cytometry. The CT-26 cells were subcutaneous inoculated into BALB/c mice, then the BALB/c mice was immunotherapyed by intramuscular injection of recombinant adenovirus Ad5-CCL20 combined with treatment of heat (combined therapy group), and simultaneitily set hyperthermia alone heat group, Ad5-CCL20 alone group, Ad5-GFP group and control group. The tumor volumes and survival rate of tumor-bearing in each group was observed. ELISAPOT method was used to examine the IFN-γ production of spleen-derived T lymphocytes and the activity of cytotoxic T lymphocytes (CTL) was measured by lactate dehydrogenase (LDH) method. The infiltration of DC at the tumor site was examined with immunohistochemistry. Results:The CT-26 cells highly expressed chemokine CCL20 after the transfection of recombinant adenovirus Ad5-CCL20. CCL20 showed obviously chemotaxis activity both to iDC and mDC, especially iDC (P<0.05). Western blotting results showed that the expression of HSP70 and GP96 were detected in CT-26 cells after heat shock, but not in pure CT-26 cells. Compared with control and unheat group, flow cytometry demonstrated that the protein from heat-shocked CT-26 cells could significantly up-regulate expressions of CD80,CD86,CCR6 and MHC-Ⅱ molecules on the DC(P<0.01). Compared with all other groups, the growth of tumor in Ad5-CCL20/heat group was significantly inhibited (\[876.8±108.7\] vs \[2 862±85.52\], \[2 660±142.6\], \[2 447±100.6\], \[1 608±135.3\] mm3,P<0.01),and the survival time of tumor-bearing mice was prolonged(P<0.05). Results of ELISAPOT and LDH respectively showed that the CTL activity of spleen-derived T lymphocytes in Ad5-CCL20/heat group was higher as compared with all other groups (P<0.05).The immunohistochemical assay also showed that the infiltration proportion of CD11c+ DC was obviously increased in the Ad5-CCL20/heat group compared with Control group, Ad5-GFP group, Ad5-CCL20 group and Hyperthermia group(P<0.05). Conclusion: Recombinant adenovirus Ad5-CCL20 combined with hyperthermia significantly inhibited tumor growth and prolonged the survival time of tumor-bearing mice by recruiting and promoting DC maturation and antigen presentation, which could be a potential therapy for colon cancer.

上海市科委医学引导项目资助(No.124119a3602)