摘要
目的选择穿膜肽的基本序列之一，构建目的多肽序列标记异硫氰酸荧光素（FITC）及钆喷替酸葡甲胺（Gd-DTPA），探讨穿膜肽携带FITC及Gd-DTPA跨膜转运性能及MR分子显像的价值。方法在穿膜肽9聚精氨酸[arginine]基础上，构建新目的多肽序列CPP13：LAGRRRRRRRRRK，固相合成多肽后标记FITC（记为CPP13-FITC）和Gd-DTPA（记为CPP13-DTPA-Gd）；分别取CPP13-FITC和FITC溶于无血清Dulbecco最低必须培养液（DMEM）培养基中，待HEPG2细胞及鼠骨髓干细胞爬片上生长至80％-90％汇合时，取2只30mm^2皿，弃去培养皿中培养液，一皿中加入CPP13-FITC／DMEM溶液，另一皿中加入FITC／DMEM溶液，37℃CO2孵箱中抚育10、30、60min时依次取出1片，磷酸平衡盐溶液（PBS）冲洗爬片后置于倒置荧光显微镜上观察细胞内出现荧光素分布的时间和部位。CPP13-DTPA-Gd作用肝癌细胞株HEPG2后，MRI研究CPP13-DTPA-Gd在细胞内转运性能及MRI的特点，将CPP13-DTPA-Gd溶于无血清DMEM培养基中，浓度为3mg／ml。待HEPG，细胞在100mm。培养皿中长至80％-90％汇合时，取3只皿分别加入CPP13-DTPA-Gd的DMEM溶液、DTPA-Gd／DMEM溶液和DMEM溶液，孵育30min后弃去培养液，以0．1MPBS反复冲洗，胰酶消化，加入2m 1l％琼脂糖／PBS溶液混匀，装入2ml离心管中，将3管固定于装有150ml 1％琼脂糖／PBS溶液的微量加样枪头盒中，待凝固后测定MR信号。结果固相合成多肽成功，测定分子量为1792．78，与理论值接近。纯度达到95％以上；标记荧光素后，倒置荧光显微镜观察细胞摄取显示10min时肝癌细胞株HEPG2和鼠骨髓干细胞胞质与细胞核内均出现荧光分布；CPP13标记的Gd-DTPA，作用细胞30min后MRI显示CPP13-DTPA-Gd作用HEPG2细胞组呈短TI、短T2信号。3层面内3组细胞感兴趣区T1信号强度（Ii）与琼脂糖T1信号强度（Io）的比值及统计学分析如下：（1）号管3层面内Iil／Io（为CPP�
Objective To study the value of a new intracellular contrast agent cell penetrating peptide labeled Fluorescein-5-isothiocyanate （FITC） and MRI contrast agent, Gadopentetate dimeglumine in molecular imaging. Methods A new cell penetration peptides （CPPs） sequence LAGRRRRRRRRRK were synthesized in solid phase on the base of arginine and were labelled with FITC （ CPP13-FITC ） and Gd - DTPA （CPP13-DTPA-Gd）. Hepatic carcinoma cell line-HEPG2 and mouse bone marrow stem cell was respectively stained by CPP13-FITC for different time intervals for observing the uptake and intracellular distribution. HEPG2 in threel00 mm2 culture plates was respectively incubated with CPP13-DTPA-Gd, Gd- DTPA and Dulbecco minimum essential medium for 30 min and imaged by 1.5 T MRI for studying the intracellular uptake and T1WI signal characteristics. Results The peptide was synthesized by the manual solid-phase method successfully . The calculated molecular weight was 1792. 78 and the chemical purity was over 95%. By inverted fluorescence microscope , HEPG2 and mouse stem cell could transport CPP-FITC in cytoplasm and nuclear in 10 min. By MR imaging , CPP-DTPA-Gd could be uptake by HEPG2 in 30min and had a short T1 short T2 signal, furthermore. T1WI signal intensity ratio between in-tube （ Ii ） and out-tube （Io） in three groups of three scan slices were shown below： Iil/Io of group 1 （Group 1 was the cell incubated by CPP13-DTPA-Gd ） respectively was 2. 84,2.60,2. 48 ; Iil/Io of group 2 （ Group 2 was the cell incubated by DTPA-Gd） respectively is 1.15,1.11,1.12 ; Iil/Io of group 3 （ Group 3 was the controled cell ） respectively was 1. 15,1.11,1.11. By ANVOA analysis ,the signal intensity among group 1,group 2 and group 3 had significant difference（F（1,2） =201.88 P 〈0. 001 , F（1,3） =206. 37 P 〈0. 001 ） meanwhile, the signal intensity between Group 2 and Group 3 had no difference （ F（2,3）= 0. 529 P = 0. 507 ）. Conclusion The new constructedcell penetration peptide on the base of
出处
《中华放射学杂志》
CAS
CSCD
北大核心
2006年第2期139-143,共5页
Chinese Journal of Radiology
基金
西安交通大学博士论文基金资助项目(dfxjtu2005-09)
关键词
肽类
磁共振成像
分子显像
造影剂
荧光素-5-异硫氰酸盐
Peptides
Magnetic resonance imaging
Molecular imaging
Contrast media
Fluorescein-5-isothiocyanate
