短双歧杆菌携带内皮抑素和干扰素γ的特性及其抗小鼠肺癌的作用

Features of Bifidobacterium breve carrying ES and IFNγ and its anti-tumor effect in mice with lung cancer

目的:以短双歧杆菌为载体,以内皮抑素(ES)和干扰素γ(IFNγ)为目的基因,探讨携带目的基因的短双歧杆菌在小鼠体内的分布特性及其抗小鼠肺癌的效果。方法:构建原核表达载体pNZ44-IFNγ及pNZ44-ssEndostatin,电穿孔法将质粒转入用3H-TdR标记的短双歧杆菌,筛选鉴定。制备荷瘤小鼠,实验分为对照组(Control)、双歧杆菌无质粒组(B-b)、双歧杆菌空质粒组(B-b-pNZ44)、双歧杆菌内皮抑素组(B-b-pNZ44-ssEndostatin)、双歧杆菌干扰素组(B-b-pNZ44-IFNγ)和双歧杆菌联合组(B-b-pNZ44-ssEndostatin+B-b-pNZ44-IFNγ),通过尾静脉注射和灌胃给予携带目的基因的短双歧杆菌。观察短双歧杆菌在小鼠体内的分布情况,以及移植瘤体积、小鼠免疫学指标和肿瘤间质微血管密度(IMVD)的变化。结果:成功构建了携带目的基因的短双歧杆菌。经尾静脉注射后,实验动物的心脏、肝脏、肺脏和肾脏中双歧杆菌逐步被清除,从第3天开始,各器官中放射活性较第1天明显降低(P<0.05);肿瘤组织的放射活性随时间延长逐渐增加,从第3天开始放射活性与第1天比较差异有统计学意义(P<0.05)。通过灌胃和尾静脉注射携带目的基因的短双歧杆菌,能抑制小鼠肺癌移植瘤的生长,并且增加CTL细胞活性、NK细胞活性和TNFα分泌活性,降低IMVD。结论:携带ES和IFNγ的短双歧杆菌能够靶向性地在肿瘤组织中定植,并通过免疫增强和抑制血管生成等机制起到抗肿瘤作用。

Objective To study the features of Bifidobacterium breve carrying endostatin（ES）and interferon γ（IFNγ）and its anti-tumor effect in mice with lung cancer.Methods The target genes ES and IFNγ were transferred into Bifidobacterium breve by plasmids and identified,then the Bifidobacterium breve marked by 3H-TdR were given into lung-cancer-bearing mice by caudal vein injection and by gavage.The experiment included control,Bifidobacterium breve without plasmid（B-b）,Bifidobacterium breve with empty plasmid（B-b-pNZ44）,Bifidobacterium breve with endostatin（B-b-pNZ44-ssEndostatin）,Bifidobacterium breve with IFNγ（B-b-pNZ44-IFNγ） and Bifidobacterium breve conbination（B-b-pNZ44-ssEndostatin ＋ B-b-pNZ44-IFNγ） groups.The distribution of the Bifidobacterium breve,the changes of tumor size,the activity of splenic cytotoxic T cell（CTL）,natural killer cell（NK）,tumor necrosis factor-α（TNF-α） secretion activity and intratumoral micro-vessel density（IMVD） were detected.Results The Bifidobacterium breve carrying ES and IFNγ were successfully constructed,and accumulated inside of the tumor.The radioactivities of different organs were significantly decreased from the 3rd day compared with the 1st day（P〈0.05）;the radioactivities of tumor tissues were significantly increased with the prolongation of time from the 3rd day compared with the 1st day（P〈0.05）.The administration of Bifidobacterium breve carrying ES and IFNγ surpressed the growth of tumor,enhanced the activities of CTL,NK and the TNFα secretion activity and reduced the IMVD.Conclusion Bifidobacterium breve carrying ES and IFNγ can targetly implant in tumor and have anti-tumor effect by immune enhancement and vessel growth inhibition.