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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 Based on the prevalence of anaerobic growth of hypoxia and Clostridium sporogenes in solid tumors, Clostridium sporogenes was used to deliver IL-12 to the tumor site. The anti-tumor effect and its toxicity were observed. Methods IL-12 cDNA was cloned into Escherichia coli-anaerobic shuttle plasmid pIMP1 by genetic engineering and transformed into Clostridium sporogenes. The expression of IL-12 was detected by ELISA and Western blot. The biological activity of IFN-γ was tested by IFN-γ induction. The mouse tumor model was made by using mouse breast cancer cell EMT6. The anti-tumor effect was observed after the tail vein was injected with Clostridium difficile Its toxicity. Results ELISA and Western blot showed that Clostridium sporogenes secreted IL-12 and co-cultured with mouse spleen cells in vitro could induce the release of IFN-γ. After intravenous injection of Clostridium sp. Clostridium into tumor-bearing mice, the serum IFN-γ was significantly increased and the tumor atrophy. No toxic effects such as diarrhea, weight loss and death associated with IL-12 were observed during the experiment. Conclusion IL-12 produced by Clostridium sporogenes has anti-mouse mammary tumor effect with no obvious toxicity.