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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7.94×108IU/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 recombinant adenovirus serotype 5/35 (Ad5 / 35) containing human melanoma-associated antigen 3 (MAGE-A3) and to observe the effect of adenovirus-mediated MAGE- Effect of A3 over-expression on maturation and apoptosis of dendritic cells in melanoma patients. Methods: The adenoviral vector with the highest transfection efficiency was selected to construct a recombinant adenovirus vector and adenovirus particles Ad5 / F35-MAGE-A3. The effect of Ad5 / F35-MAGE-A3 infection on the expression of MAGE-A3 in DCs of healthy people, patients with renal cell carcinoma and melanoma patients was detected by immunohistochemistry and Western blotting. The expression of Ad5 / F35-MAGE-A3 was detected by flow cytometry Effect of melanoma on DC maturation and apoptosis. Results: The recombinant adenovirus vector containing human MAGE-A3 was successfully constructed and adenovirus particles Ad5 / F35-MAGE-A3 were packaged. The virus titer was 7.94 × 108 IU / ml. Infection with Ad5 / F35-MAGE-A3 significantly increased the expression of MAGE-A3 (P <0.05), and did not affect the expression of MAGE-A3 in DCs in melanoma patients [(0.3352 ± 0.1272 vs (0.4672 ± 0.0704), P> 0.05]. The CD costimulatory molecules 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)%] were significantly higher than those in the negative control group and the blank (P <0.05). However, there was no significant difference in the rate of DC apoptosis between the two groups ([1.18 ± 0.09]% vs (1.09 ± 0.11)%, P> 0.05]. CONCLUSION: Recombinant adenovirus vector Ad5 / F35-MAGE-A3 can efficiently transfect melanoma DCs without affecting the expression of MAGE-A3 in DCs. It can promote the maturation of DCs without obvious cytotoxicity.