Toll样受体7激动剂T7-利尿酸偶合物联合ROR1具有更强的抗乳腺癌作用  

Combination of TLR7 agonist T7-ethacrynic acid conjugate with ROR1 has a stronger anti-breast cancer effect

作　　者：张娜[1] 靳广毅[2] 晋贞超 刘兵[2] 彭博雅[2] 高宁宁[2] 胡云龙[2] 唐俐[1] 

机构地区：[1]重庆医科大学病理生理学教研室,组织工程与干细胞研究室,重庆400016 [2]深圳大学医学部,广东深圳518060

出　　处：《细胞与分子免疫学杂志》2016年第7期876-880,885,共6页Chinese Journal of Cellular and Molecular Immunology

基　　金：国家自然科学基金(81272571);重庆市渝中区科技计划项目(20120208)

摘　　要：目的探讨Toll样受体7激动剂T7-利尿酸偶合物(T7-EA)联合受体酪氨酸激酶样孤儿受体1(ROR1)抗乳腺癌的作用。方法采用Syfpeithi表位预测软件预测ROR1的细胞毒性T淋巴细胞(CTL)表位;ELISA检测4μmol/L T7-EA、4μmol/L ROR1和4μmol/L T7-EA联合4μmol/L ROR1诱导小鼠脾淋巴细胞产生的γ干扰素(IFN-γ)和白细胞介素12(IL-12)和骨髓来源的树突状细胞(DC)产生的肿瘤坏死因子α(TNF-α);BALB/c小鼠皮下接种乳腺癌4T1细胞建立移植瘤模型,腹腔分别注射3 mg/kg T7-EA、15 mg/kg ROR1、3 mg/kg T7-EA联合15 mg/kg ROR1,每周1次,免疫治疗4次后处死小鼠检测肿瘤质量;ELISA检测血清中针对4T1细胞肿瘤蛋白的Ig G水平;乳酸脱氢酶(LDH)法测定特异性CTL活性。结果在预测的多条ROR1 CTL表位肽中选取序列PYCDETSSV作为疫苗多肽;体外实验显示T7-EA活化淋巴细胞呈现剂量依赖性,与ROR1相关多肽混合后不改变其活性;T7-EA与ROR1合用诱导小鼠脾淋巴细胞产生IFN-γ和IL-12,刺激DC产生TNF-α;体内实验显示T7-EA联合ROR1抑制肿瘤生长效果显著强于单用T7-EA组和ROR1组,T7-EA联合ROR1诱导的针对4T1细胞肿瘤蛋白的血清Ig G水平显著高于T7-EA和ROR1单独给药组,T7-EA联合ROR1诱发的T细胞杀伤作用显著强于T7-EA和ROR1单独处理组。结论 T7-EA和ROR1联合应用具有更强的抗乳腺癌效果。Objective To investigate the synergistic anti-breast cancer effect of Toll-like receptor 7 agonist T7-ethacrynic acid conjugate （T7-EA） in combination with receptor-tyrosine-kinase-like orphan receptor 1 （ROR1）. Methods ROR1 cytotoxic T lymphocyte （CTL） epitope was predicted using Syfpeithi online software. Mouse spleen lymphocytes and bone marrow dendritic cells （DCs） were separately stimulated with 4 μmol/L. TT-EA and 4 μmol/L ROR1 alone or in combination. ELISA assay was used to measure the levels of interferon-γ （IFN-γ）, interleukin 12 （IL-12） and tumor necrosis factor-α（ （TNF-α）. Xenograft model was established via subcutaneous injection of mouse breast cancer 4T1 cells. The mice were weekly treated through intraperitoneal administration of 3 mg/kg T7-EA, t5 mg/kg ROR1 or the combination of T7-EA and ROR1. After four rounds of treatment, tumor tissues were weighed. Serum level of anti-4T1 tumor protein IgG was measured by ELISA. Specific CTL activity was detected by lactate dehydrogenase （LDH） assay. Results The peptide PYCDETSSV was chosen as an antigen epitope of breast cancer. The T7-EA highly activated in vitro lymphocytes in a dose-dependent manner, which wasn＇t affected by other relevant peptides. The combination of T7-EA and ROR1 stimulated the secretion of IFN-γ and IL-12 by lymphocytes and TNF-α by bone marrow DCs. The growth of tumor in vivo was significantly inhibited by T7-EA combined with ROR1 compared with T7-EA or ROR1 alone. The specific CTL activity triggered by T7-EA combined with ROR1 was much stronger than that triggered by T7-EA or ROR1 alone. The titer of anti-4T1 tumor protein IgG induced by T7-EA combined with ROR1 was higher than that induced by T7-EA or ROR1. Conclusion The combination of T7-EA and ROR1 has a better killing effect on breast cancer.

关 键 词：乳腺癌 TOLL样受体7 T7-利尿酸偶合物(T7-EA) 受体酪氨酸激酶样孤儿受体1(ROR1) 肿瘤免疫治疗 

分 类 号：R392.9[医药卫生—免疫学] R737.9[医药卫生—基础医学]