基质金属蛋白酶MMP-2/9分子探针对临床结肠癌组织的检测  

A fluorogenic probe for sensitive detection of MMP-2/9 activities in clinical colorectal cancer tissues

作　　者：余祖红 栾富娟[1] 冷霞 史玉雪 王洁[1] 尹玲 何蕾[2,3] 史海斌 陈卫昌[1] 

机构地区：[1]苏州大学附属第一医院消化内科,苏州215006 [2]苏州大学医学部放射医学与防护学院,苏州215123 [3]苏州大学放射医学及交叉学科研究院(RAD-X),苏州215123

出　　处：《科学通报》2018年第3期318-326,共9页Chinese Science Bulletin

基　　金：国家重点研发计划(2016YFC0101200); 国家自然科学基金(81272737,21572153); 江苏省高校自然科学研究重大项目(15KJA310004); 苏州市科技计划(SYG201520); 江苏省科技厅临床专项(BL2014046)资助

摘　　要：研发基质金属蛋白酶MMP-2/9特异性识别的荧光分子探针并用于临床结肠癌组织的快速、准确性检测意义重大,但仍然具有一定的挑战性.本文基于基质金属蛋白酶MMP-2和MMP-9(MMP-2/9)特异性识别的多肽底物,利用一价铜离子催化的"点击"化学和酰胺化反应,分别将荧光素(FITC-N_3)和荧光淬灭基团Dabcyl-NHS偶联于多肽底物两端,制备得到MMP-2/9特异性识别的荧光能量共振转移(FRET)荧光分子探针Dab-GPLGVRGYFITC.通过将重组基质金属蛋白酶MMP-2/9与探针作用,跟踪检测溶液在520 nm处荧光信号变化的情况,对探针的特异性和检测灵敏度进行评估.此外,利用人源大肠癌细胞LoVo和30例临床结肠癌组织样本开展了结肠癌快速体外检测初步研究.体外重组酶检测实验表明,探针对MMP-2/9具有较强的特异性和选择性,对MMP-2的最低检测限可达14 ng/mL.共聚焦显微成像实验显示,相比于正常人胃黏膜上皮细胞GES-1,肠癌细胞LoVo表现出较强的绿色荧光信号,如预先加入抑制剂,细胞的荧光信号则很弱.另外,15例活性MMP-2高表达的临床结直肠癌组织裂解液与探针作用后荧光信号较胃炎组织增强5~9倍,而15例癌旁组织裂解液荧光则增强约0.9~3.0倍.值得注意的是,如果将探针喷洒到结肠癌组织和癌旁组织表面,相比于胃炎组织,荧光信号明显增强.总之,该探针对基质金属蛋白酶MMP-2/9过表达肠癌细胞和结肠癌组织表现出较好的特异性和检测灵敏度,具有应用于临床结肠癌早期诊断的极大潜力.Developing a novel fluorogenic probe which can be specifically recognized by active matrix metalloproteinase-2 and matrix metalloproteinase-9（MMP-2/9） to rapidly and accurately detect the clinical colorectal cancer is significant but remains a great challenge. Herein, we have designed and synthesized a novel fluorescence resonance energy transfer（FRET）-based probe Dab-GPLGVRGY-FITC for specific detection of MMP-2/9 activity. The probe was prepared by conjugating fluorescein-N_3（FITC-N_3） and fluorescent quencher dabcyl-NHS to MMP-2/9-responsive peptide substrate GPLGVRGY through copper（I）-catalyzed ＂click＂ and amidation reactions, respectively. To assess the specificity and sensitivity of the probe for active MMP-2/9 in vitro, the enzymatic assays were firstly performed with recombinant MMP-2/9. Fluorescent signal change of the mixed solution of probe and MMP-2 with and without treatment of MMP-2 specific inhibitor was recorded at 520 nm with fluorescence spectrometer. The capability of probe for specific detection of endogenous MMP-2/9 activity was further evaluated with human colorectal cancer cell Lo Vo and 30 clinical colorectal cancer tissue samples. The in vitro enzymatic assay results indicated that the probe showed good specificity and sensitivity for recombinant MMP-2/9 comparing to other proteins. The detection limit for recombinant MMP-2 is estimated to be 14 ng m L-1. Besides, the confocal laser scanning microscopy（CLSM） imaging results showed that enhanced green fluorescence signals were collected for Lo Vo cells upon treatment with probe. However, most of the fluorescence was readily competed away by pretreatment of the probe with inhibitor. In sharp contrast, only weak fluorescence was observed for normal gastric mucosa epithelial GES-1 cells. Additionally, the lysates of 15 clinical colorectal tissues displayed ~5 to 9-fold fluorescence enhancements after treatment of the probe in comparison with normal gastritis tissues. However, the fluorescence intensities of 15 peficanc

关 键 词：荧光探针 荧光能量共振转移(FRET) 基质金属蛋白酶MMP-2/9 结肠癌 

分 类 号：R735.35[医药卫生—肿瘤]