【摘要】 目的构建以基质金属蛋白酶2为靶点的穿膜肽,标记荧光素及顺磁性粒子,初步探讨体外成像价值。方法固相合成基质金属蛋白酶2（MMP-2）为靶点的可活化穿膜肽（CPPs）,标记荧光素 FITC,构建荧光素分子探针 A;标记磁共振对比剂二亚乙基三胺五乙酸钆（Gd-DTPA）,构建磁共振分析探针 B。高效液相色谱纯化探针及质谱测定分子量。聚丙烯酰胺等电聚焦电泳测定可活化穿膜肽的等电点。400 MHz 磁共振谱仪翻转恢复序列测定磁共振探针 B 的自旋-点阵弛缓时间（T₁）;制备人肺癌细胞株 A549爬片,FITC 和荧光素分子探针 A 分别孵育细胞爬片30 min 倒置荧光显微镜观察细胞内荧光分布;抗 MMP-2单克隆抗体孵育细胞后,荧光素分子探针 A 孵育细胞后荧光显像及流式细胞分析细胞摄取的状况。明胶酶谱分析细胞表达 MMP-2的情况;以 A549为对象,120nmol/ml 磁共振探针 B 和常规磁共振对比剂分别作用于细胞10、30、60、90 min 后,1.5T MRI T₁WI 视觉评价法分析细胞内磁共振信号,并与空白细胞组比较,方差分析各组细胞信号变化特征。透射电镜分析磁共振探针 B 细胞内分布。结果采用标准的芴甲氧羰基合成方案,质谱鉴定荧光素分子探针A分子量3789.74;磁共振分子探针 B 分子量3911.93。测定聚丙烯酰胺等电聚焦电泳目的条带 pH值为11.005,即为可活化穿膜肽的等电点。17℃400 MHz 磁共振谱仪测定浓度为0.5 mmol/L 的 Gd-DTPA 与磁共振探针 B 去离子水溶液的纵向驰豫时间 T₁分别为0.052 s±0.01 s 和0.050 s±0.001 s,两种配合物自旋-晶格驰豫效能分别为4.998 L·mmol^-·s^-1及6.452 L·mmol^-1·s^-1。倒置荧光镜成像显示 FITC 作用细胞组内未见荧光素分布,荧光素探针 A 作用组 A549细胞质和细胞核内出现明显得荧光素分布。抗 MMP-2单克隆抗体孵育 A549后,荧光摄取减少。以明胶酶谱分析细胞无血清培养基中 MMP-2表达显示肿瘤表达酶原和活性酶两种形式。120 nmoL/ml 磁共振探针 B 和常规磁共振对比剂分别作用于 A549 10、30、60、90 min 后,MR FSE T₁WI 显示,磁共振探针 B 作用A549细胞组呈短 T₁高信号,Gd-DPTA 作用组与对照组呈等 T₁信号。各组细胞组感兴趣区信号与背景信号比值的方差分析显示,磁共振分子探针 B 作用五组细胞信号间差异有统计学意义（F=267.569,P<0.001）,两两组间信号差异也存在统计学意义（P<0.05）,随着孵育时间增长,信号强度逐渐增强,到90 min 时未见到明显饱和现象出现。Gd-DTPA 作用细胞组信号与空白对照细胞组差异无统计学意义（P>0.05）。透射电镜显像示磁共振探针作用的肺癌细胞株 A549胞质及胞核里出现高电子密度的钆颗粒沉积,证实肿瘤细胞摄取磁共振探针 B。结论以 MMP-2为靶点的可活化穿膜肽,能够被活性基质金属蛋白酶激活,能够标记荧光探针和磁共振探针用于肿瘤细胞显像。更多还原

【Abstract】 Objective To study invasively imaging MMP2-positive tumor cell by paramagnetic Gadolinium or fluorescein carried by a activable cell penetrating peptides.Methods To label Fluorescein- 5-isothiocyanate（FITC）and gadopentetate with the activable cell-penetrating peptides by a solid-phase synthesis method.Identification by TOF mass spectrum（TOF-MS）.Isoelectrie point of the activable cell penetrating peptides is determined by disc electrophoresis.T1 relaxion of gadopentetate labeled with the activable cell-penetrating peptides（B）in water were determined on 400 MHZ NMR.Human lung cancer cell lines:A549 were respectively stained by FITC labeled with ACPPs（A）or FITC alone.MMP2 expression and activity were determined by zymography.T1WI signal of A549 incubated with 120 nmol/ml B or Diethylenetriaminepentaaeetic Acid-Gadolinium（Gd-DTPA）for different times were obtained by 1.5T MRI.The location of B in A549 was detected by Transmission Electron Microscopy.Visualization analysis and half-quantitative analysis were used to determine the signal characteristics.The ANOVA analysis and the paired samples t test were performed by SPSS 13.0.Results MALDI TOF-MS molecular weigh of A and B respectively is 3789.74 and 3911.93.Isoelectric point is 11.005T1 Relaxion of 0.5 mmol/L Gd-DTPA and B at 17 ℃ respectively is（0.052±0.01）see and（0.050±0.001）sec.Fluorescein uptake assays showed that A transloeated into A549 but would be inhibited by MMP2 antibody.Zymography showed both actived- MMP2（67000）and pro-MMP2（72000）expressed byA549.MR imaging showed A549 incubating with B had a high T₁ signal,and the signal of A549 incubating with Gd-DTPA is similar with that of the control group.Furthermore,ANOVA suggested that the T₁ signal intensity of A549 incubating with B was effected by incubating time（F=267.569,P<0.001）and increasing in a time-dependent fashion at the observed time.There is no defferenee between the T₁ signal intensity of A549 incubating with Gd-DTPA and the control group（P>0.05）.TEM showed A in cytoplasm and nucleus.Conclusion The study in vitro suggests that the MMP-2 activable cell-penetrating peptides bearing contrast media can detect the MMP2- oositive tumor cell更多还原