IL-12修饰的生孢梭菌抗小鼠乳腺癌作用观察  

Therapeutic effect of Clostridium sporogenes modified with IL-12 on murine tumors

作　　者：张艳丽[1] 张文卿[1] 王秋波[1] 丁守怡[1] 

机构地区：[1]青岛大学医学院病原生物学实验室,山东青岛266071

出　　处：《中国病原生物学杂志》2014年第7期621-624,632,共5页Journal of Pathogen Biology

基　　金：山东省自然科学基金项目(No.ZR2012HQ005);山东省医药卫生科技发展计划项目(No.2009HD005)

摘　　要：目的基于实体瘤内部普遍存在缺氧区及生孢梭菌厌氧生长的特性,利用生孢梭菌靶向递送IL-12至肿瘤部位,观察其抗肿瘤效果及其毒副作用。方法利用基因工程技术将IL-12cDNA克隆至大肠埃希菌-厌氧菌穿梭质粒pIMP1并转入生孢梭菌。用ELISA和Western blot分析其IL-12的表达;IFN-γ诱导试验测定其生物学活性;利用小鼠乳腺癌细胞EMT6制备小鼠肿瘤模型,尾静脉注射重组梭菌后观察其抗肿瘤效果及其毒性表现。结果 ELISA和Western blot显示重组生孢梭菌可分泌IL-12,体外与小鼠脾脏细胞共同培养可以引起IFN-γ的释放。重组生孢梭菌静脉注射到荷瘤小鼠体内后,小鼠血清IFN-γ显著增高,肿瘤萎缩。实验过程中未观察到与IL-12相关的腹泻、体重减轻和死亡等毒性作用。结论利用重组生孢梭菌生产的IL-12具有抗小鼠乳腺肿瘤作用,且没有明显毒性。Objective To utilize Clostridium sporogenes as a vector to deliver IL-12 to tumor sites in a targeted manner and to observe its antitumor action and toxicity in light of the hypoxia prevalent in solid tumors and the anaerobic growth characteristics of C.sporogenes Methods L-12 cDNA was cloned into an E.coli-clostridia shuttle plasmid pIMP1 by genetic engineering,and the plasmid was transformed into C.sporogenes（ATCC 3584-rhIL-12）via electroporation.ELISA and Western blotting were used to analyze the expression of IL-12.An IFN-γinduction assay was used to determine the biological activity of the secreted IL-12 in vitro.A mouse tumor model was created by subcutaneously injecting mouse mammary carcinoma cells,EMT6,into the right chest of mice.After intravenous injection（108 CFU in 0.1ml of PBS injected via the tail vein）,the antitumor action and the toxicity of the recombinant C.sporogenes were investigated.Results The results of ELISA indicated that the average level of IL-12 in the supernatant of recombinant C.sporogenes was as high as 16.5pg/ml.Western blotting revealed a target band that was detectible with goat anti-human IL-12 polyclonal antibody in the supernatant of recombinant C.sporogenes but not in that of wild-type C.sporogenes.When cultured with mice splenocytes in vitro,the recombinant C.sporogenesinduced splenocytes to release IFN-γto a level of up to 70.13pg/ml.On day 13 after intravenous injection,the IFN-γlevel in mouse plasma increased markedly in the group injected with recombinant C.sporogenes（128.55pg/ml）,and this level differed significantly from that in the group injected with wild-type C.sporogenes（65.41pg/ml）and in the PBS control（40.36pg/ml）（F=123.4,P〈0.01）.Tumors in the group injected with recombinant C.sporogenes shrank markedly,resulting in an average tumor volume of 810.88mm3 on day 20.The tumor volume in this group differed significantly from that in the group injected with wild-type C.sporogenes（5 510.18mm3）and in the PBS control（4 153.34mm3）（F=

关 键 词：生孢梭菌 IL-12 pIMP1 抗肿瘤效果 毒性 

分 类 号：R378.8[医药卫生—病原生物学]