表达可溶性程序性死亡受体1溶瘤腺病毒对B16／F10细胞增殖凋亡的影响及其机制

Effects and mechanism of recombinant oncolytic adenovirus expressing soluble programmed cell death protein 1 on proliferation and apoptosis of B16/F10 cell

目的构建表达可溶性程序性死亡受体1（sPD1）溶瘤腺病毒并研究其对B16/F10细胞增殖凋亡影响及机制。方法构建表达sPD1溶瘤腺病毒并感染B16/F10细胞株，荧光定量聚合酶链反应（PCR）技术对比分析病毒复制功能，噻唑蓝（MTT）法检测重组病毒抑制B16/F10生长；注射病毒于B16/F10皮下瘤小鼠模型中，观察肿瘤体积变化，EliSpot技术检测肿瘤浸润淋巴细胞数量。结果病毒处理B16/F10细胞6、24、36、48、60、72 h后，Ad5处理组病毒拷贝数增加倍数分别是0.99±0.10、1.12±0.05、1.57±0.15、3.59±1.03、6.26±0.74、9.69±1.46；Ad5-sPD1处理组病毒拷贝增加倍数分别是1.00±0.104、0.99±0.08、1.05±0.03、3.56±1.04、6.04±0.38、11.05±1.12，两组比较差异无统计学意义（P=0.754）；分别使用不同病毒量，感染复数（MOI）为0、5、10、15、20的病毒处理B16/F10细胞72 h后，Ad5处理组细胞活力为（100.00±6.39）%、（101.60±5.21）%、（98.66±7.31）%、（93.50±4.54）%、（73.16±2.45）%；Ad5-sPD1处理组细胞活力为（100.00±6.38）%、（97.10±6.37）%、（96.96±7.44）%、（95.67±7.46）%、（74.49±4.14）%，两组比较差异无统计学意义（P=0.779）；Mock（无病毒感染）、Ad5和Ad5-sPD1病毒治疗B16/F10皮下瘤小鼠模型，实验终止时，肿瘤体积分别是（3 638±1.823）、（2 603±336）、（1 097±532） cm^3，差异有统计学意义（Mock和Ad5：P=0.044，Mock和Ad5-sPD1：P=0.000，Ad5和Ad5-sPD1：P=0.000）。Mock、Ad5和Ad5-sPD1病毒分别注射小鼠模型，5 d后，γ-干扰素（IFN-γ）斑点数分别是19.4±7.75、149.8±25.18、314±32.34，各组间差异有统计学意义（Mock和Ad5：P=0.000，Mock和Ad5-sPD1：P=0.000，Ad5和Ad5-sPD-1：P=0.000）。结论表达sPD1溶瘤腺病毒通过阻断免疫抑制通路，增加肿瘤特异性淋巴细胞浸润，提高溶瘤腺病毒的抗肿瘤作用，抑制肿瘤生长。

ObjectiveTo construct a recombinant oncolytic adenovirus expressing soluble programmed cell death protein 1 （sPD1） and investigate its effects and mechanism on proliferation and inhibition of B16/F10 cells.MethodsThe recombinant oncolytic adenovirus Ad5-sPD1 was constructed, and infected into B16/f10 cells. The quantitative fluorescent polymerase chain reaction was used to detect the viral replication. MTT assay was applied to examine the inhibition of B16/F10 cells. The viruses were injected subcutaneously into the mouse B16/F10 tumor model to study the antitumor effect of Ad5-sPD1 in vivo. EliSpot technique was exployed to test the number of tumor proliferative lymph nodes.ResultsAfter viral treatment of B16/F10 cells for 6, 24, 36, 48, 60 and 72 h, the copies fold increases in the Ad5 treatment group were respectively 0.99±0.10, 1.12±0.05, 1.57±0.15, 3.59±1.03, 6.26±0.74, 9.69±1.46, and those in the Ad5-sPD1 treatment group were respectively 1.00±0.104, 0.99±0.08, 1.05±0.03, 3.56±1.04, 6.04±0.38, 11.05±1.12. There was no significant difference between the two groups （P=0.754）. After treating B16/F10 cells for 72 h with multiplicity of infection （MOI） of 0, 5, 10, 15 and 20, the cell viability of Ad5 treatment group was respectively （100.00±6.39）%, （101.60±5.21）%, （98.66±7.31）%, （93.50±4.54）%, （73.16±2.45）%, and that in the Ad5-sPD1 treatment group was respectively （100.00±6.38）%, （97.10±6.37）%, （96.96±7.44）%, （95.67±7.46）%, （74.49±4.14）%. There was no significant difference between the two groups （P=0.779）. After treating mice model of B16/F10 subcutaneous tumor with Ad5 and Ad5-sPD1 viruses, tumor volume was respectively 3 638±1.823, 2 603±336 and 1 097±532 at the termination of the experiments with the difference being significant （Mock and Ad5： P=0.044; Mock and Ad5-sPD1： P=0.000; Ad5 and Ad5-sPD1： P=0.000）. After respectively injecting mice model of B16/F10 subcutaneous tumor with Mock, Ad5 and Ad5 -sPD1 viruses, the number of IFN-γ spots was respectively 19.4±7.75, 149.8±25.18 and 314±32.34 with the the difference being significant （Mock and Ad5： P=0.000; Mock and Ad5-sPD1： P=0.000; Ad5 and Ad5-sPD1： P=0.000）.ConclusionRecombinant oncolytic adenovirus expressing PD1 gene can significantly improve the anti-tumor effect of the oncolytic adenovirus by blocking PD-1/PD-L1 inhibitory pathway.