局部应用CpG寡聚核苷酸联合4-1BB单克隆抗体治疗小鼠肝癌荷瘤的研究

Study of local application of CpG-oligodeoxynucleotide combined with 4-1BB monoclonal antibody to treat hepatoma- bearing mice

目的探讨局部应用CpG寡聚核苷酸(CpG-ODN)联合4-1BB单克隆抗体对小鼠肝癌的疗效,并评价4-1BB单克隆抗体对CpG-ODN免疫治疗效果的影响。方法在BALB/c雄鼠四肢腋窝皮下注射H22单细胞悬液,建立荷瘤小鼠模型,7 d后筛选出30只荷瘤体积相当的小鼠,随机分成模型对照组、CpG组和CpG+4-1BB组,并在左下肢瘤内注射药物。选取同批正常小鼠作为正常对照组。记录小鼠生存情况,计算荷瘤体积、脏器指数,检测血清白细胞介素(IL)-12和干扰素(IFN)γ浓度以及脾脏CD8+T淋巴细胞比例。计量数据用方差分析,各组小鼠存活率采用Log-Rank检验分析。结果处死前模型对照组小鼠荷瘤持续增长,CpG组和CpG+4-1BB组荷瘤体积[(976.08±29.55)mm3、(47.25±0.93)mm3)]与模型组[(1 336.52±39.40)mm3]相比均减小(F = 5 329.273,P < 0.05);CpG+4-1BB组远处荷瘤体积[(611.83±113.02)mm3]与模型组和CpG组[(1 406.62±51.09)mm3、(1 380.01±51.44)mm3]相比减小(F = 247.160,P < 0.05),但CpG组与模型组无明显差异(P > 0.05)。模型组血清IL-12浓度[(23.90±2.33)pg/ml]、IFNγ浓度[(103.02±6.10)pg/ml]和脾脏CD8+T淋巴细胞比例(4.54%±0.62%)较正常组均减低(P值均< 0.05);CpG组和CpG+4-1BB组血清中IL-12浓度[(29.21±2.23)pg/ml、(37.04±1.49)pg/ml]、IFNγ浓度[(116.12±4.08)pg/ml、(138.65±1.72)pg/ml)、CD8+T淋巴细胞比例(6.65%±0.64%、12.73%±0.88%)均高于模型组(P值均< 0.05);CpG+4-1BB组高于CpG组(P < 0.05)。CpG+4-1BB组小鼠存活率均高于模型组和CpG组(χ2 = 25.544,P < 0.05),CpG组与模型组之间无明显差异(P > 0.05)。四组脏器指数无明显差异(P > 0.05)。结论4-1BB单克隆抗体联合CpG-ODN治疗可使肝癌荷瘤体积缩小,并可抑制远处荷瘤增长,明显延长小鼠存活时间。

Objective To investigate the curative effect of local application of CpG-oligodeoxynucleotide (CpG-ODN) combined with 4-1BB monoclonal antibody in hepatoma-bearing mice, and to evaluate the effect of 4-1BB monoclonal antibody on CpG-ODN immunotherapy. Methods H22 single cell suspension was injected subcutaneously into the axilla and four limbs of the BALB/c male mice to establish a tumor-bearing mice model. After 7 days, 30 mice with corresponding tumor-bearing volume were screened and randomly divided into model control group, CpG group and CpG+4-1BB group, and the drug was injected into the tumors of left lower extremity. The same batch of normal mice was selected as normal control group. Survival of mice was recorded. Tumor-bearing volume and organ index were calculated. Serum levels of interleukin (IL)- 12 and interferon (IFN) gamma and spleen CD8+T lymphocyte ratio were measured. The measurement data were analyzed by analysis of variance. The survival rate of each group of mice was analyzed by log-rank test. Results Mice in the model control group with tumor-bearing volume had a sustained growth before the execution. CpG group and the CpG+4-1BB group [(976.08 ± 29.55) mm3,(47.25 ± 0.93) mm3)] tumor-bearing volume was decreased than model group [(1 336.52 ± 39.40) mm3](F = 5 329.273, P < 0.05). CpG+4-1BB group distant tumor-bearing volume [(611.83 ± 113.02) mm3] was decreased than model group and CpG group [(1 406.62 ± 51.09) mm3,(1 380.01 ± 51.44) mm3](F = 247.160, P < 0.05), but there was no significant difference between the CpG group and the model group (P > 0.05). Serum IL-12 concentration (23.90 ± 2.33 pg/ml), IFN-γ concentration (103.02 ± 6.10 pg/ml) and spleen CD8+T cell ratio (4.54 ± 0.62%) in the model group were lower than those in the normal group (P < 0.05). Serum IL-12 concentration in CpG group and CpG+4-1BB group (29.21 ± 2.23 pg/ml, 37.04 ± 1.49 pg/ml), IFN-γ concentration (116.12 ± 4.08 pg/ml, 138.65 ± 1.72 pg/ml), CD8+T cell ratio (6.65 ± 0.64%, 12.73 ± 0.88%) were higher than the model group, while CpG+4-1BB group was higher than the CpG group (P < 0.05). The survival rate of CpG+4-1BB group was higher than that of model group and CpG group (χ2 = 25.544, P < 0.05), but there was no significant difference between CpG group and model group (P > 0.05). There was no significant difference in organ index between the four groups (P > 0.05). Conclusion 4-1BB monoclonal antibody combined with CpG-ODN therapy can shrink hepatoma-bearing capacity, inhibit the growth of distant tumors and significantly prolong the survival time of mice.