以葡萄糖调节蛋白78(GRP78)为靶向的胰蛋白酶抑制剂抗肠癌效应研究  

Anti-cancer effects of trypsin inhibitor targeting glucose-regulated protein 78(GRP78) in colorectal cancer cells

作　　者：赵超[1] 李宗伟[1] 付荣[1] 赵亚蕊[1] 史通麟[1] 李卓玉[1] 

机构地区：[1]山西大学生物技术研究所-化学生物学与分子工程教育部重点实验室,太原030006

出　　处：《复旦学报（医学版）》2013年第2期148-152,158,共6页Fudan University Journal of Medical Sciences

基　　金：国家自然科学基金项目(31201072;31271516;81150026);山西省回国留学人员科研资助项目(2010-2012);山西省青年科学基金项目(2012021028-4);山西省国际科技合作项目(2011081058)~~

摘　　要：目的构建以肿瘤细胞表面葡萄糖调节蛋白78(glucose-regulated protein 78,GRP78)为靶向的重组绿豆胰蛋白酶抑制剂(trypsin inhibitor,TI)活性片段GBP-TI,并探讨其抗肠癌的分子靶向效应。方法用带有GRP78结合肽(GRP78binding peptide,GBP)表达序列的引物PCR扩增胰蛋白酶抑制剂活性片段,构建GBP-TI的原核表达载体;采用GST亲和层析柱纯化获得GBP-TI蛋白;激光共聚焦方法检测GBP-TI与GRP78的相互作用;MTT和DAPI细胞核染色方法分别检测GBP-TI对大肠癌细胞存活和凋亡的影响。结果 GBP-TI能够与大肠癌细胞表面的GRP78相互作用;与GST-TI相比,GBP-TI能够剂量依赖性地诱导大肠癌细胞死亡,而对正常细胞生长无明显影响。结论 GBP-TI可以通过结合肿瘤细胞表面GRP78蛋白特异性杀伤肿瘤细胞。本研究为肿瘤靶向治疗提供了一种新的策略。Objective To construct the active fragment of recombinant trypsin inhibitor GBP-TI,which targets glucose-regulated protein 78（GRP78） existed in tumor cell surface,and to investigate its molecular targeting effects on colorectal cancer cells.Methods To construct the GBP-TI expressing vector,the primer containing the expressed sequence of GRP78 binding peptide（GBP） was used to amplify the trypsin inhibitor active fragment（TI）;GBP-TI protein was purified by GST-affinity chromatography.The interaction of GBP-TI with GRP78 on cell surface was confirmed by confocal immunofluorescence analysis.The effects of GBP-TI on cell survival and apoptosis were detected by MTT and DAPI staining,respectively.Results GBP-TI was able to interact with GRP78 existed in colorectal cancer cell surface.In contrast to GST-TI,GBP-TI could induce apoptosis in colorectal cancer cells in a dose-dependent manner,but had a negligible effect on the growth of normal cells.Conclusions GBP-TI can kill tumor cells through specifically binding to cell surface GRP78.This study provides a new strategy for targeted cancer therapy.

关 键 词：胰蛋白酶抑制剂(TI) 葡萄糖调节蛋白78(GRP78) 大肠癌 靶向药物 

分 类 号：R735.3[医药卫生—肿瘤]