口服携带Tip30与人IFN-γ基因减毒沙门菌治疗舌癌荷瘤裸鼠的实验研究

Gene therapy for tongue carcinoma mediated by oral administration of attenuated Salmonella harboring tip30 and IFN-γ genes in nude mouse model

目的:观察口服携带HIV tip30与人IFN-γ基因减毒沙门菌(SL7207)基因治疗舌癌荷瘤裸鼠的疗效。方法:4周龄BALB/c雄性裸鼠25只,随机分为PBS对照组、单纯SL7207组、携带人IFN-γ基因SL7207-pCI-IFN治疗组、携带HIV tip30基因SL7207-pCI-tip30治疗组和同时携带HIV tip30与人IFN-γ基因SL7207-pCI-tip30/IFN联合治疗组5组。裸鼠下颌下区皮下接种人舌癌Tca8113细胞10 d后,口服相应治疗菌,每周1次,共3次。测定瘤体积及瘤重变化,计算抑瘤率,免疫组化检测肿瘤组织中IFN-γ、Tip30的表达,琼脂糖凝胶电泳检测细胞凋亡。采用SPSS15.0软件包对数据进行统计学分析。结果:肿瘤生长曲线、动物累积生存率及肿瘤抑瘤率指标均显示,PBS对照组肿瘤呈进行性生长;单纯SL7207组显示一定抑瘤效果(P<0.05);携带人IFN-γ基因SL7207-pCI-IFN治疗组和携带HIV tip30基因SL7207-pCI-tip30治疗组均具有较好疗效(P<0.05),但2组之间无显著差异;Tip30和IFN联合SL7207-pCI-tip30/IFN治疗组疗效最好(P<0.01)。表明Tip30和IFN联合具有协同抗肿瘤作用。免疫组化检测发现,口服携带不同目的基因重组菌的肿瘤组织内相应蛋白高表达。琼脂糖凝胶电泳分析发现,转tip30基因的肿瘤细胞基因组DNA表现出凋亡DNA"阶梯状"条带。结论:口服重组减毒沙门菌具有体内瘤体转基因的能力,而且可使同时携带的HIV tip30与人IFN-γ2个目的基因同时达到瘤体细胞内表达,发挥协同抗瘤作用,为舌癌的基因治疗提供了新的方法。

PURPOSE： To observe the effect of gene therapy mediated by oral administration of attenuated Salmonella carrying tip30 and IFN-r/genes in human tongue carcinoma nude mouse model. METHODS： 25 four-week- old BALB/C male nude mice were divided randomly into 5 groups based on the differently harboring genes in the recombinant attenuated Salmonella typhimuriums SL7207, which included blank group （PBS）, control group （SL7207）, IFN treatments group （SL7207-pCI-IFN）, tip30 treatment group （SL7207-pCI-tip30） and the combination of IFN and tip30 treatment group （SL7207-pCI-tip30/IFN）. On 10d after submandibular subcutaneous injection of Tca8113 cells into the mice, the recombinant attenuated Salmonella typhimuriurns were orally administrated 3 times at 1 week interval. The treatment effect indicators included the growth curve, the tumor inhabitation rate, the survival rate, the apoptosis typical DNA ladder and the protein expressions of tip30 and IFN-~/ in tumor cells. Statistical analysis was performed with SPSS15.0 software package. RESULTS： The growth curve, the tumor inhabitation rate and the survival rate indicated that the tumor in the PBS group constantly grew up and induced the animal death in earlier time, while the SL7207 treatmenthad a slight inhibiting effect compared with PBS group. However, the SL7207-pCI-IFN group and the SL7207-pCI-tip30 had a moderate inhibiting effect compared with the SL7207 group, while the combination of IFN and tip30 had the strongest inhibiting effect. The protein expressions of tip30 and IFN-r were detected in tumor cells while the typical DNA ladders of apoptosis were observed only in the tip30 gene transfer groups （SL7207-pCI-tip30 and SL7207-pCI-tip30/ IFN）. CONCLUSIONS： The recombinant attenuated Salmonella typhimurium possesses a capacity of gene transfer in vivo through oral administration. The combination of the harboring tip30 and IFN-r genes have synergistic inhibiting tumor effect for tongue carcinoma. Supported by National Natural Science Foundation of China（30572060）.