PD-1抗体联合CXCL10对肝癌的抑制作用及其机制研究

Combination therapy with anti-PD-1 and CXCL10 against hepatocellular carcinoma

目的:使用腺病毒为载体,联合PD-1抗体和趋化因子CXCL10,探讨其对HepG2细胞的生长抑制作用及其机制。方法:PCR、酶切、连接等技术构建pAd-AFP-anti-PD-1-CXCL10腺病毒表达载体,经测序验证其正确性。Western blot检测蛋白anti-PD-1单链抗体及CXCL10的表达。ELISA检测CXCL10的分泌水平。流式细胞术检测活化T细胞表面CXCR3表达变化,及与T细胞共培养后HepG2表面PD-L1表达。Transwell方法研究趋化因子CXCL10对活化T细胞趋化的影响。建立HepG2裸鼠皮下移植瘤模型,检测腺病毒pAd-AFP-anti-PD-1-CXCL10的体内抑瘤效果。结果:蛋白anti-PD-1及CXCL10表达正确,CXCL10约(1.35±0.33)ng/ml,T细胞活化后CXCR3表达升高,与HepG2共培养可上调其表面PD-L1表达。腺病毒pAd-AFP-anti-PD-1-CXCL10表达的CXCL10可显著趋化T细胞,体内可抑制HepG2肿瘤生长。结论:免疫检查点抑制剂联合趋化因子,募集T细胞并解除免疫抑制,为肿瘤的靶向治疗提供了新思路。

Objective:Using adenovirus as a carrier and combining anti-PD-1 antibody with CXCL10 to explore its antitumor effect on HepG2 cells.Methods:The expression vector of adenovirus(pAD-AFP-anti-PD-1-CXCL10)was constructed by PCR,enzyme digestion,and ligation.The correctness of these constructs was identified by DNA sequencing.The expression of anti-PD-1 single-chain antibody and CXCL10 was detected by western blot.The secretion level of CXCL10 was detected by ELISA.The expression of CXCR3 on the activated T cells and the expression of PD-L1 on the surface of HepG2 after co-culture with T cells was detected by flow cytometry.The migration ability of activated T cells towards CXCL10 was determined by transwell.Subcutaneous-orthotopic transplantation was used to build the HepG2 orthotopic hepatocarcinoma model in athymic Balb/c mice,and the antitumor effect of adenovirus pAd-AFP-anti-PD-1-CXCL10 in vivo was measured.Results:The expression of protein anti-PD-1 and CXCL10 was confirmed.The concentration of CXCL10 was about 1.35±0.33 ng/mL.The expression of CXCR3 was up-regulated after T cell activation.Co-cultivation with HepG2 could up-regulate the expression of PD-L1.CXCL10 expressed by adenovirus pAd-AFP-anti-PD-1-CXCL10 could significantly recruit T cells.This therapeutic method partly inhibited HepG2 tumor growth in vivo.Conclusions:Immune checkpoint inhibitors combined with chemokines could recruit T cells and relieving immunosuppression,providing a new idea for targeted tumor therapy.