乳腺癌特异性多肽介导生物大分子体内外效应  

Effect of breast-cancer-specific peptide-mediated protein transduction in vivo and in vitro

作　　者：耿计伟[1] 高嫦娥[1] 刘为青[1] 熊秋霞[1] 缪延栋[1] 周华华[1] 董坚[1] 

机构地区：[1]昆明医学院第一附属医院生物治疗中心,昆明650032

出　　处：《科学通报》2012年第10期813-820,共8页Chinese Science Bulletin

基　　金：国家自然科学基金(3086033);云南省应用基础研究重点项目(2009CC023);云南省科技条件平台建设(2007DA006)资助

摘　　要：探讨乳腺癌特异性多肽PI携带外源性生物大分子靶向抗肿瘤的作用.将分离纯化获得的融合蛋白PI-EGFP与靶细胞MDA-MB-231体外共培养,探讨融合蛋白与靶细胞的结合能力;将此融合蛋白经尾静脉及肿瘤局部注射入荷瘤裸鼠体内,探讨其在肿瘤部位的聚集程度;利用分子克隆技术构建重组原核表达载体pET-28a(+)-pI-tk,诱导表达、分离纯化、鉴定获得的融合蛋白PI-HSV-TK,将不同浓度的融合蛋白与MDA-MB-231细胞共培养,经更昔洛韦(ganciclovir,GCV)作用后,探讨PI-HSV-tk对细胞的靶向杀伤效应.荧光显微镜下观察,在靶细胞内可检测到绿色荧光信号,经尾静脉及局部注射融合蛋白PI-EGFP后,在不同组织器官可见不同强度的荧光信号,在尾静脉注射组的肾脏和肿瘤部位可检测到荧光信号,而局部注射组仅在肿瘤部位可检测到;成功构建了重组原核表达载体pET-28a(+)-pI-tk;分离纯化获得高效表达的PI-TK融合蛋白,SDS-PAGE电泳及Western blotting鉴定融合蛋白的表达正确;CCK-8法及流式细胞仪检测显示,GCV对转导融合蛋白的MDA-MB-231细胞有杀伤作用,IC50值为152.64μg/mL.乳腺癌特异性转导多肽能携带生物大分子进入靶细胞,并携带具有杀伤效应的物质,使其发挥靶向治疗作用,为进一步探讨该多肽作为靶向性载体奠定实验基础和理论依据.To investigate the effects of a breast-cancer-specific peptide（PI）-enhanced green fluorescent protein（EGFP） fusion protein in vivo and the PI-HSV-tk/GCV system in vitro,the in vitro transduction efficiency of PI-EGFP was tested in MDA-MB-231 and MDA-MB-435 cells.Mice were also injected with PI-EGFP via the tail vein and its distribution in relation to local tumors was observed in vivo.The recombinant plasmid pET-28a（＋）-PI-tk was constructed and the fusion protein PI-HSV-tk was then detected in MDA-MB-231 cells by sodium dodecyl sulfate-polyacrylamide gel electrophoresis（SDS-PAGE） and Western blotting.Cell Counting Kit-8（CCK-8） and fluorescence-activated cell sorting were used to determine the killing effect of ganciclovir（GCV） on MDA-MB-231 cells.Green fluorescence could be detected in MDA-MB-231 cells following transduction with PI-EGFP.Green fluorescence could also be detected in tumors,but not in other tissues and organs,after injection via the tail vein.The recombinant plasmid pET-28a（＋）-PI-tk was constructed successfully and purified PI-tk fusion protein was confirmed by SDS-PAGE and Western blotting.CCK-8 analysis indicated the cell-killing effect of GCV on MDA-MB-231 cells after transduction with the fusion protein,with an IC 50 value of 152.64 μg/mL.PI could effectively transduce biological macromolecules into target cells and thus produce a cell-killing effect with potential for targeted therapy.PI displays good targeting efficiency in MDA-MB-231 tumor cells.This study provides experimental evidence to support further investigations into this peptide as a targeting vector.

关 键 词：乳腺癌 融合蛋白 靶向治疗 增强型绿色荧光蛋白(EGFP) 单纯疱疹病毒胸腺嘧啶核苷激酶(HSV-tk) 

分 类 号：Q71[生物学—分子生物学]