目的：构建含人黑素瘤相关抗原3（melanoma-associated antigen 3，MAGE-A3）的重组5/35型腺病毒（adenovirus serotype 5/35，Ad5/F35），观察腺病毒介导的MAGE-A3过表达对黑素瘤患者树突状细胞（dendritic cell，DC）的成熟和凋亡的影响。方法：选择转染效率最高的腺病毒载体，构建重组腺病毒载体并包装腺病毒颗粒Ad5/F35-MAGE-A3。免疫组化和Western blotting检测Ad5/F35-MAGE-A3感染对健康人、肾癌患者及黑素瘤患者DC中MAGE-A3表达的影响，流式细胞术检测Ad5/F35-MAGE-A3感染对黑素瘤患者DC成熟和凋亡的影响。结果：成功构建了含人MAGE-A3的重组腺病毒载体并包装腺病毒颗粒Ad5/F35-MAGE-A3，病毒感染滴度为7.94×108 IU/ml。Ad5/F35-MAGE-A3感染显著提高了健康人和肾癌患者DC中MAGE-A3的表达（P<0.05），不影响黑素瘤患者DC中MAGE-A3的表达\[（0.3352±0.1272）vs（0.4672±0.0704），P>0.05\]。Ad5/F35-MAGE-A3感染后黑素瘤患者DC共刺激分子CD80\[（20.42±0.58）% vs（10.22±1.04）%、（8.95±02）%\]、CD86\[（85.3±3.98）% vs（39.85±2.86）%、（34.1±4.32）%\]和HLA-DR\[（86.87±4.36）% vs（63.68±3.15）%、（60.69±4.81）%\]的表达明显高于阴性对照组和空白对照组（均P<0.05），但DC凋亡率无显著差异\[（1.18±0.09）% vs （1.09±0.11） %，P>0.05\]。结论：重组腺病毒载体Ad5/F35-MAGE-A3能够高效转染黑素瘤患者的DC，感染后不影响MAGE-A3在DC中的表达，能够促进DC的成熟，无明显细胞毒作用。

Objective:To construct a recombinant adenovirus Ad5/F35 containing human melanoma-associated antigen 3 (MAGE-A3), and to observe the effect of adenovirus-mediated MAGE-A3 overexpression on the maturation and apoptosis of dendritic cells (DCs) in patients with melanoma. Methods: To choose the adenoviral vectors with the highest transfection efficiency, and then to construct recombinant adenovirus vectors and packaging adenovirus particles Ad5/F35-MAGE-A3. Immunohistochemistry and Western blotting were performed to detect the influence of Ad5/F35-MAGE-A3 infection on the expression of MAGE-A3 of DCs in healthy people and patients with kidney cancer or melanoma. Flow cytometry was used to detect the effect of Ad5/F35-MAGE-A3 infection on the maturation and apoptosis of DCs in patients with melanoma. Results: The recombinant adenovirus vector containing human MAGE-A3 was successfully constructed and adenovirus particles Ad5/F35-MAGE-A3 were packaged with the infective titer of 7.94×108 IU/ml. Ad5/F35-MAGE-A3 infection improved the MAGE-A3 expression of DCs in healthy people and kidney cancer patients (P<0.05), and it did not affect MAGE-A3 expression of DCs in melanoma patients (\[0.3352±0.1272\] vs \[0.4672±0.0704\], P＞0.05). After Ad5/F35-MAGE-A3 infection, the co-stimulatory molecule CD80 (\[20.42±0.58\]% vs \[10.22±104\]%, \[895±0.2\]%), CD86 (\[85.3±3.98\]% vs \[39.85±2.86\]%, \[34.1±4.32\]%) and HLA-DR (\[8687±436\]% vs \[63.68±3.15\]%, \[60.69±4.81\]%) which expressed on the surface of DCs was significantly higher than that of the negative control group and the blank control group (all P<0.05), but no significant difference existed in apoptosis rate (\[1.18±0.09\]% vs \[1.09±0.11\]%, P>0.05).Conclusion: Recombinant adenovirus vector can efficiently affect DCs. Ad5/F35-MAGE-A3 infection may not affect the expression of MAGE-A3 of DCs in melanoma patients, and promote the maturation of DCs without obvious cytotoxicity.

“十一五”新药创制重大专项资助项目（No. 2009zx09503）