rmhTNF-α联合PD-1单抗治疗小鼠Lewis肺癌的研究

rmhTNF-α联合PD-1单抗治疗小鼠Lewis 肺癌的研究作者：朱辽辽张存徐盈李晓菊高源韩俊张英起来源：《中国医药导报》2020年第10期[摘要] 目的研究重組改构人肿瘤坏死因子-α（rmhTNF-α）联合PD-1单抗（anti-PD-1）对小鼠Lewis肺癌的治疗效果及作用机制。

方法 24只6～8周龄C57BL/6雌鼠，依据随机数字表法分为4组，每组6只：生理盐水组、rmhTNF-α组、anti-PD-1单抗组、联合给药组。

游标卡尺测瘤体并用活体成像观测。

16 d后安乐处死小鼠，剥离肿瘤称重。

观察小鼠生存期。

免疫组化检测肿瘤CD8+T细胞数目。

结果与生理盐水组比较，rmhTNF-α组、anti-PD-1组、联合给药组肿瘤体积、瘤重均减小，差异有高度统计学意义（P < 0.01）;rmhTNF-α组肿瘤体积大于联合给药组，差异有统计学意义（P < 0.05）;而联合给药组与anti-PD-1组肿瘤体积差异无统计学意义（P > 0.05）;联合给药组瘤重分别与rmhTNF-α、anti-PD-1组比较，差异均无统计学意义（均P > 0.05）。

与生理盐水组比较，rmhTNF-α组、anti-PD-1组、联合给药组生存期增加，差异有统计学意义（P < 0.05或P < 0.01），而联合给药组生存期分别与rmhTNF-α组、anti-PD-1组比较，差异均无统计学意义（均P > 0.05）。

rmhTNF-α组与生理盐水组比较，肿瘤CD8+T细胞数目增多（P < 0.05）;anti-PD-1组、联合给药组与生理盐水组比较，肿瘤CD8+T细胞数目显著增加（P < 0.01）;联合给药组肿瘤CD8+T细胞数目少于rmhTNF-α组（P < 0.01），而与anti-PD-1组比较，肿瘤CD8+T细胞数目差异无统计学意义（P > 0.05）。

结论rmhTNF-α具有抑制小鼠肺癌的作用，但不具有增强PD-1抗体治疗肺癌的作用。

[关键词] 重组改构人肿瘤坏死因子-α;PD-1单抗;CD8+T细胞;Lewis肺癌[中图分类号] R730.2 ; ; ; ; ;[文献标识码] A ; ; ; ; ;[文章编号] 1673-7210（2020）04（a）-0004-04Study of rmhTNF-α combin ed with anti-PD-1 in the treatment of Lewis lung cancer in miceZHU Liaoliao ; ZHANG Cun ; XU Ying ; LI Xiaoju ; GAO Yuan ; HAN Jun ; ZHANG YingqiBiopharmaceutical Teaching and Research Office， Department of Pharmacy， Air Force Medical University， Shaanxi Province，Xi′an ; 710032， China[Abstract] Objective To study the therapeutic effect and mechanism of recombinant mutated human tumor necrosis factor-α （rmhTNF-α） combined with PD-1 monoclonal antibody （anti-PD-1） in Lewis lung cancer in mice. Methods A total of 24 female C57BL/6 mice aged 6 to 8 weeks were divided into 4 groups according to the random number table method， with 6 mice in each group： normal saline group， rmhTNF-group， anti-PD-1 group， and combined administration group. The tumor was measured with vernier caliper and observed by in vivo imaging. After 16 days， the mice were euthanized and the tumor was removed and weighed. The survival period of mice were observed. Immunohistochemical was used to detect tumor CD8+T cells. Results Compared with the normal saline group， the tumor volume and tumor weight in the rmhTNF-α group， anti-PD-1 group and the combined administration group all decreased， with highly statistically significant differences （P < 0.01）. The tumor volume of rmhTNF-α group was larger than that of the combined administration group， and the difference was statistically significant （P < 0.05）. However， there was no significant difference in tumor volume between the combined administration group and the anti-PD-1 group （P > 0.05）. There was no statistically significant difference in tumor weight between the combined administration group and the rmhTNF-α group and anti-PD-1 group （all P > 0.05）. Compared with the normal saline group， the survival of rmhTNF-α grou p， anti-PD-1 group and the combined administration group increased， and the differences were statistically significant （P < 0.05 or P < 0.01）， while the survival of the combined administration group was not statistically significant compared with the rmhTNF-α group and anti-PD-1 group （all P > 0.05）. Compared with the normal saline group， the number of CD8+T cells increased in the rmhTNF-α group （P < 0.05）. Compared with the normal saline group， the number of CD8+T cells increased significantly in the anti-PD-1 group and combined administration group （P < 0.01）. The number of CD8+T cells in the combined administration group was less than that in the rmhTNF-α group （P< ;0.01）， while the number of CD8+T cells was not statistically significant compared with that in the anti-PD-1 group （P > 0.05）. Conclusion RmhTNF-α can inhibit lung cancer in mice， but it can not enhance the effect of anti-PD-1 to treat lung cancer.[Key words] Recombinant mutated human tumor necrosis factor-α; PD-1 monoclonal antibody; CD8+T cell; Lewis lung cancer肿瘤坏死因子-α（TNF-α）由巨噬细胞产生，介导内毒素诱导的肿瘤坏死[1]，在免疫中发挥关键作用[2]。

TNF-α可以改变血管通透性，使抗肿瘤药物更易到达肿瘤组织周围[3]。

免疫检查点抑制剂PD-1单抗（anti-PD-1）治疗晚期肺癌有效率为20%，与细胞因子的联合治疗成为新策略[4-5]。

因此，本研究利用优化的重组改构人肿瘤坏死因子-α（recombinant mutated human tumor necrosis factor-α，rmhTNF-α）联合anti-PD-1治疗小鼠Lewis肺癌，探索rmhTNF-α是否具有提高anti-PD-1治疗效果的作用及作用机制。

1 材料与方法1.1 实验材料rmhTNF-α（上海赛达生物药业有限公司，批号：SC20180401）;anti-PD-1（西安壮志生物科技有限公司，批号：BE0146）;Luciferase慢病毒（上海吉凯基因化学技术有限公司，批号：LPK001）;Lewis肺癌细胞（中国科学院上海细胞库，目录号：TCM7）;小鼠CD8分子免疫组化试剂盒（北京中杉金桥有限公司，批号：SP9000）。

1.2 实验动物24只C57BL/6雌鼠，6～8周龄，体重（18±2）g，购自空军军医大学动物中心，生产许可证号：SCXK（军）2017-0021。