微波消融联合消融灶周边注射CpG ODN诱导抗小鼠肝癌免疫效应

Microwave ablation combined with peritumoral injection of CpG ODNinduced antitumor immunity in a murine liver cancer model

目的:探讨消融灶周围注射CpG ODN诱导抗肿瘤免疫反应的效能。方法:建立C57BL/6J小鼠Hepa 1-6细胞双侧皮下肝癌模型,根据右侧皮下肝癌治疗的方法分为联合治疗组(微波消融联合消融灶周边注射CpG ODN治疗)、消融治疗组、CpG ODN治疗组和对照组。观察双侧皮下肝癌体积变化、小鼠生存时间。免疫组化检测右侧肿瘤组织热休克蛋白70(HSP70)表达和左侧肿瘤组织CD4、CD8T淋巴细胞的数量。ELISA法检测各组小鼠血清IL-12、IL-10含量。LDH释放法检测各组脾源性CTL杀伤活性。另选取单侧皮下肝癌小鼠,分别给予联合治疗和消融治疗,30天后在对侧皮下再次接种同种肝癌细胞,观察再接种肿瘤的成瘤率和肿瘤体积变化。结果:双侧皮下肝癌模型小鼠联合治疗组和消融治疗组右侧肿瘤体积体积显著小于CpG ODN治疗组和对照组(P<0.05)。联合治疗组中左侧未治疗的肿瘤体积在第10天后明显小于其他各组(P<0.05)。中位生存时间分别为:联合治疗组73天,消融治疗组60天,CpG ODN治疗组55天,对照组38天。联合治疗组左侧肿瘤组织内CD8T淋巴细胞数量为(25.17±5.46)/HP,显著高于其他3组(P<0.05)。联合治疗组和消融治疗组右侧肿瘤组织HSP70标记指数分别为(39.95±9.03)%和(33.90±7.98)%,明显高于CpG ODN治疗组和对照组(P<0.05)。联合治疗组小鼠血清IL-12含量显著高于其他各组(P<0.05),IL-10含量显著低于其他各组(P<0.05)。联合治疗组中脾CTL细胞对Hepa 1-6杀伤率显著高于其他各组(P<0.05)。单侧皮下肝癌小鼠联合治疗组再接种肿瘤细胞的成瘤率为58.33%,而消融治疗组的成瘤率为83.33%,且联合治疗组的再接种肿瘤体积在第20天后明显小于消融治疗组(P<0.05)。结论:微波消融能促使肿瘤细胞HSP70表达,微波消融联合消融灶周边注射CpG ODN能激活小鼠体内的CD8 T淋巴细胞,促使小鼠体内发生Th2/Th1免疫偏移,从而产生抗肿瘤的免疫效应,抑制肿瘤生长。

Objective：To investigate the efficacy of microwave ablation combined with peritumoral injected CpG ODN in inducing immunity response against mouse liver cancer.Methods：Bilateral liver cancer model was established by subcutaneous injection Hepa 1-6 cells on both sides of the C57BL/6J mouse abdomen.According the therapy method to the right side tumor,the mice were divided into four groups：combination therapy group（microwave ablation combined with peritumoral injected CpG ODN）,ablation therapy group,CpG ODN therapy group,and control group.Bilateral tumor volume and survive time were observed.Heat shock protein 70（HSP70） in right tumor tissue and CD4＋/CD8＋ T lymphocytes in left tumor tissue were measured by immunohistochemistry.Concentration of IL-12 and IL-10 in serum were detected by ELISA method.Splenic cytotoxic T lymphocytes（CTL） was assayed by LDH method.Moreover,unilateral subcutaneous liver cancer mice were given combination therapy or ablation therapy,Hepa 1-6 cells were rechallenged at the opposite flank after 30 days later,the tumor emerging rate and tumor volume at the opposite flank were observed.Results：In the bilateral tumor model,the right tumor volumes in combination therapy group and ablation therapy group were smaller than those in CpG ODN therapy group and control group,left tumor volumes in combination therapy group were smaller than those in other group.The median survive time in combination therapy group,ablation therapy group,CpG ODN therapy group,and control group were 73 days,60 days,55 days,38 days respectively.In the left tumor tissue,the number of CD8＋ T lymphocytes in combination therapy group were（25.17±5.46）,which were significantly higher than those in other groups.In the right tumor tissue,the labeling index of HSP70 in combination therapy group and ablation therapy group were（39.95±9.03）% and（33.90±7.98）% respectively,which were significantly higher than those in CpG ODN therapy group and control group.The serum concentration of IL-12 in combination therapy group was higher than those in other groups,but the serum concentration of IL10 in combination group were the lowest in the four groups.The cytotoxicities of CTL in combination therapy group were higher than those in other groups.In the unilateral subcutaneous liver cancer mice model,the tumor emerging rate of rechallenged tumor cells in combination therapy group and ablation therapy group were 83.33% and 58.33% respectively.After 20 days later,the tumor volume in combination therapy group was smaller than that in ablation therapy group.Conclusion：The expression of HSP70 in tumor cell can be improved by microwave ablation.Microwave ablation combined with peritumoral injection of CpG ODN can not only activate CD8＋ T lymphocytes,but also promote Th2/Th1 deviation,which can enhance the host antitumor immunity effect and inhibit tumor growth.