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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 anti-breast cancer effect of Toll-like receptor 7 agonist T7-diuretic conjugate (T7-EA) combined with receptor tyrosine kinase-like orphan receptor 1 (ROR1). Methods The cytotoxic T lymphocyte (CTL) epitopes of ROR1 were predicted by the Syfpeithi epitope prediction software. The expression of CTL epitopes in ROR1 cells was detected by ELISA with 4μmol / L T7-EA, 4μmol / L ROR1 and 4μmol / L T7- Tumor necrosis factor alpha (TNF-alpha) produced by lymphocyte-derived interferon gamma (IFN-y) and interleukin 12 (IL-12) and bone marrow-derived dendritic cells (DCs); BALB / c mice The tumor model was established by subcutaneous inoculation of breast cancer 4T1 cells. The intraperitoneal injection of 3 mg / kg T7-EA, 15 mg / kg ROR1, 3 mg / kg T7-EA and 15 mg / The mice were sacrificed to test the tumor mass, Ig G level in the serum of the 4T1 cells was detected by ELISA, and CTL activity was measured by the lactate dehydrogenase (LDH) method. Results The sequence of PYCDETSSV was selected as the vaccine peptide from the predicted ROR1 CTL epitope peptides. In vitro experiments showed that T7-EA-activated lymphocytes showed a dose-dependent manner and did not change its activity when mixed with ROR1-related peptides. T7-EA combined with ROR1 T7-EA combined with ROR1 inhibited tumor growth significantly stronger than T7-EA group and ROR1 group, T7-EA group and ROR1 group In combination with ROR1, the level of serum IgG of 4T1 cell tumor protein was significantly higher than that of T7-EA and ROR1 alone groups. T7-EA and ROR1-induced T cell cytotoxicity was significantly stronger than T7-EA and ROR1 alone groups. Conclusion The combination of T7-EA and ROR1 has stronger anti-breast cancer effect.