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1

摘要

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.%0bjective:To investigate the efficacy of m icrowave ablation combined wilh peritum oral irpcted CpG ODN in inducing in- m unity response against mouse liver cancer .M eyhods:Bilateial Brer cancer modelwas established by subcutaneous inaction Hepat-6 eels on bo1h sides of 1heC57BL/6Jm ouse abdomen .A cooiding 1he Iheiapy m eihod to 1he right side 1unor,1he mice were divided into tour groups; combination Iherapy group (m icrowave ablation combined with peritiinoial injscted CpG ODN ) ,abhtion Iherapy group ,CpG ODN Iherapy group, and control group .B ilateral turn or volm e and survive tin e w ere observed .H eat shock protein 70 (H SP70 ) in right frm or tissue and CD 4 / CD 8 T ]ym phocytes in left frm or tissue w ere m easured by in m unohistochan istry .Concenttatfan of LH.2 and LH.0 in senm w ere detected by ELBA m eihod .Splenic cytotoxic T fymphocytes (CTL) was assayed by LDH meihodJI oreover,unilateral subcutaneous liver cancer mice were given con bination Iheiapy or abhtfan Iherapy ,H epa 1-6 cells w ere rechaUenged at the opposite flank after 30 days later ,1he turn or em eiging rate and tin or volume at 1he opposite flank were observed .Results; In the bilateral turn ormodel,the right tum or volm es in combination Iherapy group and ablation Iherapy group were an allerihan those in CpG ODN Iherapy group and oon1rolgroup,]eftiLinorvo]umes in combination Iherapy group were an aller 1han 1hose in oiher group .The m edian suivire tin e in conbinatfan therapy group,ablation Iherapy group ,CpG ODN Iherapy group,and control group were 73 days ,60 days ,55 days ,38 days respectively .In the left frm or tissue ,1he number of CD 8 T lymphocytes in combination Iherapy group were(25 .17+5 .46),which were sjgnificantiy higher 1han 1hose in oiher groups. In 1he right turn or tissue,the labeling index of HSP70 in combination therapy group and ablation Iheiapy group were (39 .95+9 .03 )% and (33 .90+7 .98 )% respectively,which were significantly higherihan those in CpG ODN theiapy group and oontrolgroup .The senin concenttatfan of LH.2 in ocmbination Iherapy group was higher than those in oiher groups,but the senm concentration of IL-10 in combination group were 1he hvest in 1he four groups .The cytiioxicities of CTL in combination therapy group were higher than 1hose in oiher groups. Si 1he unilateral subcutaneous Brer cancer m ice m odel,1he tun or em eiging rate of rechallenged turn or cells in can bination Iherapy group and ablation Iherapy group w ere 83 .33% and 58 .33% respective^ .A fter 20 days kter,the tun or volm e in can bination Iherapy group w as an aller 1han 1hat in ablation Iherapy group .C on- elusion :The expression ofHSP70 in turn or cell can be in proved by microwave ablation .M icrowave ablation can bined w ith perhum oral inaction of CpG ODN can not only activate CD 8 T ]ym phocytes, but also promote Th2/thl deviation,which can enhance 1he host antitin or in m unity effect and inhibit frm or grow th.