人白介素18(IL-18)插入突变体抗血管新生作用的研究  

The inhibitory effects of RGD motif containing human interleukin-18 mutant on the proliferation and adhesion of ECV304 cells

作　　者：卢忠民[1] 赵春艳[1] 任凤[1] 崔秀云[1] 赵宝昌[1] 

机构地区：[1]大连医科大学生物化学和分子生物学教研室,辽宁大连116027

出　　处：《药品评价》2005年第5期340-345,共6页Drug Evaluation

摘　　要：目的构建IL-18的定点突变体,并研究其对血管新生的抑制作用。方法利用基因重叠延伸剪接技术,在人白细胞介素18(IL-18)的cDNA序列中插入一个GGC序列,使IL-18第39位精氨酸和第40位天冬氨酸残基之间插入一个甘氨酸残基,从而构建了RGD模体。将此cDNA序列构建入表达质粒pPIC9K,并在酵母PichiaPastorisGS115中得到高效表达。结果IL-18-RGD仍保存对PBMC诱导产生IFN-γ能力外,对ECV304细胞的增殖(IC50=7.80μmol/L)和对ECV304向Fn的黏附均具有明显的抑制作用,且呈剂量依赖关系;而野生型IL-18对ECV304细胞向Fn的黏附则没有作用。结论与野生型IL-18相比,IL-18-RGD增加了抗血管生成作用,从而增强其抗肿瘤能力。Objective Study inhibitory effect of RGD motif containing human interleukin-18 mutant on angiogenesis. Methods Using site-directed mutagenesis and DNA recombinant technology, GGC fragment was inserted into human IL-18 eDNA. The mutated IL-18 eDNA was construct- ed into plasmid pPIC9K, then transformed into Pichia pastoris GS115 and efficiently expressed. A Glycine residue was inserted into the recombinant IL-18 between Arg39 and Asp40 to form a RGD motif. The mutated IL-18 was termed IL-18-RGD. The protein was purified with Sephadex G-100. Results The cell culture of ECV304 showed that IL-18-RGD and ILl8 efficiently in- hibited ECV304 proliferation, IC50 = 7.80mmol/L and 13.6mmol/L respectively. The inhibition of mutant IL-18-RGD on the adhesion of ECV304 to Fn was detected, but there is no inhibitory ef- fect of IL-18 detected. The inhibitory effect of IL-18-RGD on CAM angiogenesis. There was no differences between IL-18-RGD and wild IL-18 in the activity of inducing IFN-γ in human PBMC. Conclusions In comparision with wild IL-18, IL-18-RGD can increase the effect on anti-angiogenesis, consequently promote the ability of anti tumor.

关 键 词：人白细胞介素18 RGD模体 毕赤酵母 ECV304细胞 

分 类 号：R392[医药卫生—免疫学]