经穿膜肽与PEG修饰的核糖体失活蛋白Gelonin抗肿瘤作用的研究  

Study on cell-penetrating peptide modified and PEGylated ribosome inactive protein Gelonin

作　　者：张娅洁 王慧媛[2] 陈应之 汤懿斯 杨志民[1,4] 黄永焯[2] 

机构地区：[1]天津医科大学药学院,天津市临床药物关键技术重点实验室,天津300070 [2]中国科学院上海药物研究所,上海201203 [3]广州中医药大学热带医学研究所,广州510405 [4]美国密西根大学药学院,美国密西根州48109

出　　处：《天津医科大学学报》2015年第5期369-374,共6页Journal of Tianjin Medical University

基　　金：国家自然科学基金资助项目(81172996;81373357;81361140344;81422048)

摘　　要：目的:通过对核糖体失活蛋白Gelonin进行化学修饰,利用穿膜肽和聚乙二醇(PEG)偶联来提高其到达肿瘤部位和进入肿瘤细胞的能力,使Gelonin更高效地发挥抑瘤作用。方法:利用FPLC Superdex75分子筛预装柱纯化系统对所修饰的Gelonin进行纯化后,在不同细胞系测试细胞毒性;通过倒置荧光显微镜、流式细胞分析技术等对药物进入纤维肉瘤细胞HT1080的能力进行评价;采用小动物活体成像技术考察药物体系在荷瘤动物体内的分布情况。结果:采用分子筛色谱纯化可以得到纯度相对较高的修饰产物,其毒性较无修饰的Gelonin强,且在HT1080细胞系作用最明显;细胞摄取结果显示,与未修饰的Gelonin相比,该药物体系具有更高的细胞摄取效率;动物成像结果表明,PEG5000修饰可以改变Gelonin在动物体内的分布情况,增加在肿瘤的药物蓄积。结论:穿膜肽和PEG5000修饰后的Gelonin有较高的肿瘤细胞摄取和杀伤能力,药物在肿瘤的蓄积量较高,从而增强了药物的抑瘤效果。Objective： To improve anti-tumor effect of Gelonin, the plant-sourced RIP is modified by chemically conjugating a cell- penetrating peptide and polyethylene glycol （PEG）. Methods：Purified protein was obtained after being performed on FPLC （fast protein liquid chromatography） Superdex75 column. Cytotoxicity was detected by MTT assay. The cellular uptake by HT1080 cells was studied by using inverted fluorescence microscopy and flow cytometry. In-vivo imaging technology was utilized for investigation of the in-vivo drug distribution in the HT1080 tumor-bearing mice. Results：The modified product was purified by using gel filtration chromatergraphy. Moreover, compared with native Gelonin, the cytotoxicity of modified protein was increased, especially in HT1080, presumably due to the enhanced cellular uptake. The in-vivo imaging results suggested that drug accumulation in tumor was improved by PEGylation. Conclusion： Modified Gelonin can improve cell penetration and cytotoxicity in tumor cells. PEGylation can increase tumor accumulation of the protein drug, and thereby enhance its anti-tumor effect.

关 键 词：核糖体失活蛋白 聚乙二醇 低分子量鱼精蛋白 抗肿瘤 GELONIN 

分 类 号：R9[医药卫生—药学]